Uses of extracellular vesicle comprising a fusion protein having fc binding capacity

ABSTRACT

The present invention pertains to engineered extracellular vesicles (EVs) for use in a variety of research, diagnostic, imaging and therapeutic applications. The EVs are engineered to enable incorporation and surface display of various proteins of interest, which may be exogenous and/or endogenous in nature. The coating of EVs is achieved through inventive protein engineering of EV polypeptides, and the present invention thus relates to methods for coating of EVs, EVs per se, as well kits, detection methods, diagnostic applications, imaging and delivery methods based on said engineered EVs.

TECHNICAL FIELD

The present invention pertains to extracellular vesicles engineered tofunction as research and diagnostics tools for e.g. standardization andevaluation of assays for EV characterization, in diagnostics of variousdiseases, and as biological reference material (RM) for e.g. flowcytometry.

BACKGROUND ART

Extracellular vesicles (EVs) are nano-sized vesicles that are releasedby EV-producing cells into the extracellular environment. EVs and inparticular exosomes have been shown to be able to transport proteinbiologics, such as antibodies and decoy receptors, enabling an entirelynovel form of advanced biological therapeutics harnessing the propertiesof EVs in combination with the specificity of recombinant proteins.Moreover, the presence of different qualities of EVs dependent on theirorigin and the disease state of an organism and the subsequent value ofthe detection of specific types or qualities of EVs in terms ofdiagnostics have been demonstrated. Triggered by this plethora ofdifferent potential applications, many analysis methods aiming to size,enumerate and phenotype EVs have been established or are beingestablished. Such methods include flow cytometry, light scattering,nanoparticle tracking analysis (NTA), and/or detection of fluorescenceafter binding of different probes.

The molecular content and concentrations of EVs in human body fluidshave raised increasing interest for their use as biomarkers (Fais etal., 2016). A biomarker based on EVs has not yet been fully realized,partly due to the lack of standardization. Standardization is difficultbecause the calibration of instruments, the interpretation andvalidation of results, and the comparison of measurements require areference material (RM) with physical properties equal or very similarto the properties of EVs. One of the most analyzed properties of an EVsample is the concentration. However, the measured EV concentrationdepends on the physical properties of EVs, such as the size distributionand refractive index (RI), complicating the analysis, as explainedfurther below.

EVs smaller than 300 nm constitute the majority of EV population(Aatonen et al., 2014). Typical size distributions of EVs start at ˜30nm, exhibiting a peak at a diameter<100 nm, and follow a decreasingpower-law function or exponential function for diameters>100 nm (van derPol et al., 2016). With the exception of transmission electronmicroscopy (TEM), none of the current analytical methods are able todetect the entire population of EVs (van der Pol et al., 2016). Theinability to detect the smallest EVs leads to both differences andunderestimation of the determined concentration. Consequently, thereported number of EVs in normal human plasma ranges from 10⁴ to 10¹²mL-1 (van der Pol et al., 2014a). These 8 orders of magnitude differencein EV concentration highlights the need for standardization.

In flow cytometry, which is one of the most commonly used methods in EVstudies (Lacroix et al., 2010), particle detection is often based onlight scattering. Because the RI of silica (1.45) and polystyrene beads(1.61) is higher than the mean RI of naturally occurring EVs (˜1.39),applying a gate on the scatter signals of silica or polystyrene beadswill result in erroneous estimations of EV size and concentration (vander Pol et al., 2012, 2014b). For example, a lower size gate set with200 nm polystyrene beads, which scatter the same amount of light as EVsof ˜500 nm (Chandler et al., 2011), leads to the exclusion of EVsbetween 200 and 500 nm (van der Pol et al., 2014b). Since theconcentration of EVs decreases with increasing diameter, a polystyrenesize gate generally leads to an underestimation of the actual EVconcentration.

With nanoparticle tracking analysis (NTA) the Stokes-Einstein equationis used to derive the hydrodynamic diameter of EVs from their Brownianmotion (Dragovic et al., 2011). Although in NTA, the RI of EVs does notaffect the measured diameter, the EV size distribution and RI do affectthe measured concentration (Filipe et al., 2010), because the measuredconcentration depends on the brightness of the scattering particle.

Altogether, these examples emphasize the urgent need to develop RM witha similar RI and size distribution, but preferably also with amorphology (for TEM) and zeta potential (for tunable resistive pulsesensing, TRPS) similar to the studied EVs. Ultimately, also other RMproperties would match those of EVs, including surface molecules orinternal cargo. This is challenging because the development of anoptimal RM for EV studies and the analytical methods for their detectionare dependent on each other. Further, the different analyticaltechniques depend on different properties of EVs.

Although several studies have characterized and described the use ofmonodisperse (Lacroix et al., 2010; Chandler et al., 2011; Maas et al.,2015) and bimodal (Nicolet et al., 2016) synthetic RM, the reported useof biological RM is limited. Submicron particles with physical andbiochemical properties similar to EVs can be isolated from naturallyoccurring sources, and may include 1) isolated EV populations from, e.g.cell cultures, 2) plasma lipoproteins, plant and marine viruses, and 3)small spherically shaped (coccoid) bacteria, or picoplankton. PotentialEV sources are in vitro cell cultures, therapeutic clinical gradeerythrocyte and platelet concentrates, urine, and outer membranevesicles produced by bacteria. Here, the specific advantage is that theobtained RM have enhanced physical and biochemical similarities,including the molecular contents, with actual EVs.

Lipoproteins and viral particles from plant and marine sources aresuitable as EV-RM because they have a size distribution overlapping withthe bulk of EVs and they do have a relatively small variation in size.However, a major drawback of lipoproteins and viral particles is thatthe RI of these particles is higher than the RI of EVs, due to theirhigh protein content, a prominent problem especially ofprotein-enveloped viruses. Another issue of using viral particles istheir biosafety, which could be circumvented by producing virus-likeparticles, i.e. particles lacking the viral genome.

Biological RM can further be produced by disrupting cells to producesmall vesicles from the fragments yielding particles with varyingdiameters. The main advantage of using such materials is that thephysical and biochemical properties of the obtained RM would betterresemble EVs compared to synthetic RM. Erythrocytes are theoreticallyideal materials to use, since their cell membrane composition iswell-characterized and they are devoid of intracellular membranes.Additionally, erythrocytes are structurally stable during extendedstorage and are an easily accessible material. For these reasons,erythrocytes from clinical surplus concentrates have been used toproduce an RM for testing and evaluation and different methods forerythrocyte disruption have also been compared. Biological RM can alsobe obtained from lipid constructs such as liposomes that are extensivelyused as delivery vehicles and their production methods are well known.Liposomes of a desired size can be prepared by ultrasonication orextrusion of the starting material through e.g. polycarbonate filters ofa set pore size. An advantage of liposomes is that their RI can bemanipulated during the production.

Currently, lyophilized exosome are used as standardized positivecontrols for immunocapture performance evaluation. Lyophilization is acommonly applied technique for preserving the long-term stability ofvarious types of biological samples, and lyophilized exosomes can bepurchased as control standards for multiple applications including flowcytometry (FACS), Western blotting (WB), ELISA and as calibrationstandards for quantitation of exosome-derived markers from biologicalsamples. However, lyophilization considerably affects the biophysicaland biological properties of EVs, and as such this method for generatingbiological RM is not suitable for powerful analytical methods where thesimilarity to EVs in their natural form is required.

Thus, although extensive attempts are being made to devise relevant RMsfor EVs the field is far from arriving at a suitable solution.Furthermore, despite intense research into EVs and their ability totransport drug cargo very few useful applications of EVs as researchand/or diagnostics tools have emerged.

SUMMARY OF THE INVENTION

It is hence an object of the present invention to overcome theabove-identified problems associated with using non-biological RMs likebeads or biological RMs consisting of particles with propertiesdissimilar to those of EVs and exosomes. The present invention also aimsto satisfy other existing needs within the art, for instance to enableusing EVs as a predictable and specific positive control, as acompensation control or as a biological RM for any kind of assay thatuses e.g. antibodies, fluorescent probes, and/or various biomolecules todetect, enumerate, size or phenotype EVs, for research or diagnostics.Furthermore, the present invention also aims to provide for novelimportant uses of EVs as research tools in preclinical, clinical, anddiagnostics settings, for instance in combination withimmunohistochemistry, microscopy, radiolabeling, etc. In particular, theunique delivery properties of engineered EVs are in the presentapplication harnessed to address several obstacles within the broadfield of biomedical research.

The present invention achieves these and other objectives by utilizingFc binding polypeptides (herein often referred to as Fc binders) ofhuman and/or non-human origin to engineer the content andcharacteristics of EVs. Very often such Fc binding polypeptides arefused to exosomal polypeptide to enhance loading of EVs with the Fcbinding polypeptides. The EVs comprising Fc binding polypeptides maythen be allowed to bind to the Fc domains present on antibodies, otherproteins natively comprising Fc domains, and/or proteins that have beenmodified to comprise Fc domains for the purpose of the presentinvention.

Thus, the present invention provides for a biological RM, comprising anEV comprising at least one Fc binding polypeptide, which optionallyforms part of a fusion protein together with an exosomal polypeptide. Inan advantageous embodiment, the Fc domain of an Fc containing protein(such as an antibody, which may be tagged with a detection moiety) isbound by the Fc binding polypeptide of the EV, thereby generating acoated EV with broad utility as RM, diagnostic tool, imaging tool, andfor essentially any research purpose. In a first aspect, therefore, theinvention provides the use of an EV comprising at least one Fc bindingpolypeptide, as a research tool, a diagnostic tool, imaging tool, in adetection kit, as a biological RM, as an experimental control and/or anexperimental standard.

In another aspect, the present invention relates to an in vitro, in vivoand/or ex vivo method for delivery of an Fc containing protein to atarget cell. The method comprises the steps of (i) providing an EVcomprising at least one Fc binding polypeptide, (ii) allowing the Fcbinding polypeptide to bind the Fc containing protein, and (iii) puttingthe EV having attached to its Fc binding polypeptide the Fc containingprotein in contact with a target cell, to enable intracellular,extracellular, and/or intramembrane delivery of the Fc containingprotein in question.

In yet another aspect, the present invention provides a detection kitcomprising an EV, wherein the EV comprises a fusion protein comprisingat least one Fc binding polypeptide and at least one exosomalpolypeptide. The Fc binding polypeptide may be bound (attached) to atleast one Fc containing protein, such as an antibody. The detection kitmay be used for various purposes, for instance detection of anintracellular, intramembrane, or extracellular antigen which can beachieved by delivering e.g. a labelled antibody against the antigen inquestion.

In a further aspect, the instant invention relates to a kit of partscomprising (i) an EV comprising at least one Fc binding polypeptide, and(ii) at least one Fc containing protein. As above-mentioned, the Fcbinding polypeptide may be fused to at least one exosomal polypeptide toincrease the number of Fc binders in or on the EV.

In yet another aspect, the present invention relates to a nanoparticlecomplex between (i) an EV comprising at least one Fc binding polypeptideand (ii) at least one Fc containing protein. The at least one Fccontaining protein may be several copies of one type of Fc containingproteins (e.g. multiple copies of an antibody targeting an intracellularoncogenic protein) or more than one type of Fc containing protein (forinstance multiple copies of an antibody against one intracellular targetand multiple copies of another antibody against a membrane target).Generally, the nanoparticle complex formed between EVs as per thepresent invention and Fc containing proteins can be used broadly acrossvarious research, diagnostic and therapeutic areas, for instance withinflow cytometry, electron microscopy, confocal and/or fluorescencemicroscopy, in vitro, in vivo and/or ex vivo diagnostics, imaging and/ortreatment of target cells, and for numerous other purposes.

In further aspects, the present invention pertains to methods forproducing EVs capable of binding to proteins comprising an Fc domain.Such methods may comprise the steps of: (i) introducing into an EVsource cell a polynucleotide construct encoding a fusion proteincomprising at least one Fc binder polypeptide and at least one exosomalpolypeptide, and (ii) harvesting EVs which are secreted from the EVsource cell, said EVs comprising the fusion protein of interest.Additionally, the present invention relates to methods for coating EVswith at least one protein comprising an Fc domain, comprising the stepsof (i) providing an EV comprising a fusion protein comprising at leastone Fc binder and at least one exosomal polypeptide, and (ii) allowingthe Fc binder of the fusion protein to bind the Fc domain of at leastone protein comprising an Fc domain.

Finally, the present invention also relates to fusion proteinscomprising at least one Fc binding polypeptide and at least one exosomalpolypeptide, and polynucleotide constructs encoding for such fusionproteins, as well as vectors, EVs and cells comprising such constructs.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 . Schematic illustration of an EV comprising a fusion proteincomprising an exosomal protein fused to an Fc binding polypeptide (i.e.the Fc binder domain). The Fc binder is capable of binding e.g. anantibody and/or any other protein comprising an Fc domain, therebyturning the EV into a multivalent delivery vehicle for proteintherapeutics.

FIG. 2 . Electron microscopy pictures of EVs comprising Fc bindingpolypeptides (A) are decorated with nanogold labeled antibodies (i.e. Fccontaining proteins), whereas control EVs (B), which lack Fc bindingpolypeptides, do not have any antibodies bound to their surfaces.

FIG. 3 . Flow cytometry data showing that the EVs comprising Fc bindingpolypeptides bind Fc containing proteins of interest (human IgG). Thebinding is very efficient to all bead populations included in the kit,including the unspecific/Isotype/negative control bead populations

FIG. 4 . Anti-HER2 antibody increases uptake of antibody-decorated EVsas compared to isotype control-decorated and wild type EVs only in HER2high-expressing cell line MDA-MB-361, but not in HER2 low-expressingcell line MDA-MB-231.

FIG. 5 . Signals of fluorescently labelled antibodies are clearlypresent in cells treated with Fc-binding EVs having attached to theirsurfaces antibodies comprising Fc domains, while fluorescence signalsare absent in untreated (1) or control EV treated (2) cells, measured byfluorescence microscopy (A) and flow cytometry (B). This demonstratesthat Fc containing proteins such as antibodies can be deliveredintracellularly by Fc binding EVs, and that binding to EVs dramaticallyincreases uptake of antibodies into cells.

FIG. 6 . Successful inhibition of NFkB-mediated intracellular signalswhen an anti-NFkB antibody is delivered into cells by Fc-binding EVs(i.e. EVs comprising at least one Fc binding polypeptide fused to atleast one exosomal polypeptide).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to EVs comprising at least one Fc bindingpolypeptide for use across a large number of research, diagnostic, andtherapeutic applications. In advantageous embodiments, the at least oneFc binding polypeptide is comprised in a fusion protein with an EVpolypeptide, in order to increase the numbers of Fc binding polypeptidesin each EV. In advantageous embodiments, the Fc domain of an Fccontaining protein (such as an antibody, which may be tagged with adetection moiety) is bound by the Fc binding polypeptide of the EV,thereby generating an EV having attached to its surface the Fccontaining protein, resulting in a nanoparticle with broad utilitytechniques such as flow cytometry, nanoparticle tracking analysis,microscopy and electron microscopy, as a diagnostic tool, as an imagingtool, and for essentially any research, imaging, diagnostic, andtherapeutic purpose.

For convenience and clarity, certain terms employed herein are collectedand described below. Unless otherwise defined, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs.

Where features, aspects, embodiments, or alternatives of the presentinvention are described in terms of Markush groups, a person skilled inthe art will recognize that the invention is also thereby described interms of any individual member or subgroup of members of the Markushgroup. The person skilled in the art will further recognize that theinvention is also thereby described in terms of any combination ofindividual members or subgroups of members of Markush groups.Additionally, it should be noted that embodiments and features describedin connection with one of the aspects and/or embodiments of the presentinvention also apply mutatis mutandis to all the other aspects and/orembodiments of the invention. For example, the Fc binding polypeptidesdescribed herein in connection with the EVs comprising fusion proteinscomprising such Fc binding polypeptides are to be understood to bedisclosed, relevant, and compatible with all other aspects, teachingsand embodiments herein, for instance aspects and/or embodiments relatingto the methods for producing or coating the EVs, or relating to thepolynucleotide and polypeptide constructs described herein. Furthermore,certain embodiments described in connection with certain aspects, forinstance the use of EVs comprising Fc binding polypeptides havingattached to the EV a plurality of Fc containing proteins, as describedin relation to their use as biological RM, may naturally also berelevant in connection with other aspects and/or embodiments, forinstance embodiments pertaining to the use of EVs as diagnostic toolsand/or imaging tools in e.g. pathology and/or immuno-histochemistry.Furthermore, all polypeptides and proteins identified herein can befreely combined in fusion proteins using conventional strategies forfusing polypeptides. As a non-limiting example, all Fc bindingpolypeptides described herein may be freely combined in any combinationwith one or more exosomal polypeptides. Also, Fc binding polypeptidesmay be combined with each other to generate constructs comprising morethan one Fc binding polypeptide. Moreover, any and all features (forinstance any and all members of a Markush group) can be freely combinedwith any and all other features (for instance any and all members of anyother Markush group), e.g. any Fc binding protein may be combined withany Fc containing protein such as any antibody, or any exosomalpolypeptide may be combined with any Fc binding polypeptide.Furthermore, when teachings herein refer to EVs (and/or the EV-anchoredfusion protein—Fc containing protein complexes) in singular and/or toEVs as discrete natural nanoparticle-like vesicles it should beunderstood that all such teachings are equally relevant for andapplicable to a plurality of EVs and populations of EVs and the EVscoated with Fc containing proteins. As a general remark, the Fc bindingpolypeptides, the Fc containing proteins such as the antibodies, theEV-producing cell sources, the exosomal polypeptides, and all otheraspects, embodiments, and alternatives in accordance with the presentinvention may be freely combined in any and all possible combinationswithout deviating from the scope and the gist of the invention.Furthermore, any polypeptide or polynucleotide or any polypeptide orpolynucleotide sequences (amino acid sequences or nucleotide sequences,respectively) of the present invention may deviate considerably from theoriginal polypeptides, polynucleotides and sequences as long as anygiven molecule retains the ability to carry out the desired technicaleffect associated therewith. As long as their biological properties aremaintained the polypeptide and/or polynucleotide sequences according tothe present application may deviate with as much as 50% (calculatedusing for instance BLAST or ClustalW) as compared to the nativesequence, although a sequence identity that is as high as possible ispreferable (for instance 60%, 70%, 80%, or e.g. 90% or higher). Thecombination (fusion) of e.g. at least one Fc binding polypeptide and atleast one exosomal protein implies that certain segments of therespective polypeptides may be replaced and/or modified and/or that thesequences may be interrupted by insertion of other amino acid stretches,meaning that the deviation from the native sequence may be considerableas long as the key properties (e.g. Fc binding properties, traffickingto the surface of exosomes, binding affinity, etc.) are conserved.Similar reasoning thus naturally applies to the polynucleotide sequencesencoding for such polypeptides. All the accession numbers and SEQ ID NOsmentioned herein in connection with peptides, polypeptides and proteinsshall only be seen as examples and for information only, and allpeptides, polypeptides and proteins shall be given their ordinarymeaning as the skilled person would understand them. Thus, asabove-mentioned, the skilled person will also understand that thepresent invention encompasses not merely the specific SEQ ID NOs or theaccession numbers referred to herein but also variants and derivativesthereof. Unless mentioned otherwise, all accession numbers referred toherein are UniProtKB accession numbers as per the 22 Jun. 2017 versionof the database, and all proteins, polypeptides, peptides, nucleotidesand polynucleotides mentioned herein are to be construed according totheir conventional meaning as understood by a skilled person.

The terms “extracellular vesicle” or “EV” or “exosome” are usedinterchangeably herein and shall be understood to relate to any type ofvesicle that is obtainable from a cell in any form, for instance amicrovesicle (e.g. any vesicle shed from the plasma membrane of a cell),an exosome (e.g. any vesicle derived from the endo-lysosomal pathway),an apoptotic body (e.g. obtainable from apoptotic cells), amicroparticle (which may be derived from e.g. platelets), an ectosome(derivable from e.g. neutrophils and monocytes in serum), prostatosome(e.g. obtainable from prostate cancer cells), or a cardiosome (e.g.derivable from cardiac cells), etc. The sizes of EVs may varyconsiderably but an EV typically has a nano-sized hydrodynamic radius,i.e. a radius below 1000 nm. Clearly, EVs may be derived from any celltype, both in vivo, ex vivo, and in vitro. Furthermore, the said termsshall also be understood to relate to extracellular vesicle mimics, cellmembrane-based vesicles obtained through for instance membraneextrusion, sonication, or other techniques, etc. It will be clear to theskilled artisan that when describing research, diagnostic, medical,technical and scientific uses and applications of the EVs, the presentinvention normally relates to a plurality of EVs, i.e. a population ofEVs which may comprise thousands, millions, billions or even trillionsof EVs. As can be seen from the experimental section below, EVs may bepresent in concentrations such as 10⁵, 10⁸, 10¹⁰, 10¹¹, 10¹², 10¹³,10¹⁴, 10¹⁵, 10¹⁸, 10²⁵10³⁰ EVs (often termed “particles”) per unit ofvolume (for instance per ml), or any other number larger, smaller oranywhere in between. In the same vein, the term “population”, which maye.g. relate to an EV comprising a certain fusion protein between anexosomal polypeptide and an Fc binding polypeptide which in turn may bebound to an Fc containing protein of interest, shall be understood toencompass a plurality of entities constituting such a population. Inother words, individual EVs when present in a plurality constitute an EVpopulation. Thus, naturally, the present invention pertains both toindividual EVs and populations comprising EVs, as will be clear to theskilled person. Furthermore, the EVs of the present invention may alsocomprise additional therapeutic and/or diagnostic agents, in addition tothe Fc containing proteins which may be bound to the EV surfaces. Insome embodiments, the EV may comprise diagnostic and tracing agents suchas fluorophores, radioactive compounds, tracers, inorganic and organiccompounds, imaging agents, etc. In further embodiments, the additionaltherapeutic agent may be at least one therapeutic small molecule drug.In some embodiments, the therapeutic small molecule drug may be selectedfrom the group consisting of DNA damaging agents, agents that inhibitDNA synthesis, microtubule and tubulin binding agents, anti-metabolites,inducers of oxidative damage, anti-angiogenics, endocrine therapies,anti-estrogens, immuno-modulators such as Toll-like receptor agonists orantagonists, histone deacetylase inhibitors, inhibitors of signaltransduction such as inhibitors of kinases, inhibitors of heat shockproteins, retinoids, inhibitors of growth factor receptors, anti-mitoticcompounds, anti-inflammatories, cell cycle regulators, transcriptionfactor inhibitors, and apoptosis inducers, and any combination thereof.In further embodiments, the additional agent may be a therapeutic and/ordiagnostic and/or imaging nucleic acid-based agent. Such nucleicacid-based therapeutic agents may be selected from the group comprisingsingle-stranded RNA or DNA, double-stranded RNA or DNA, oligonucleotidessuch as siRNA, splice-switching RNA, CRISPR guide strands, short hairpinRNA (shRNA), miRNA, antisense oligonucleotides, polynucleotides such asmRNA, plasmids, or any other RNA or DNA vector. Of particular interestare nucleic acid-based agents which are chemically synthesized and/orwhich comprise chemically modified nucleotides such as 2′-O-Me,2′-O-Allyl, 2′-O-MOE, 2′-F, 2′-CE, 2′-EA 2′-FANA, LNA, CLNA, ENA, PNA,phosphorothioates, tricyclo-DNA, etc. In yet further embodiments, theEVs as per the present invention may comprise additional therapeuticand/or diagnostic agents which may be protein and/or peptides. Suchproteins and/or peptides may be present inside of the EVs, inserted intothe EV membrane or in association with the EV membrane, or may beprotruding from the EV into the extravesicular environment. Such proteinand/or peptide agents may be selected from a group of non-limitingexamples including: antibodies, intrabodies, single chain variablefragments (scFv), affibodies, bi- and multispecific antibodies orbinders, affibodies, darpins, receptors, ligands, enzymes for e.g.enzyme replacement therapy or gene editing, tumor suppressors, viral orbacterial inhibitors, cell component proteins, DNA and/or RNA bindingproteins, DNA repair inhibitors, nucleases, proteinases, integrases,transcription factors, growth factors, apoptosis inhibitors andinducers, toxins (for instance pseudomonas exotoxins), structuralproteins, neurotrophic factors such as NT3/4, brain-derived neurotrophicfactor (BDNF) and nerve growth factor (NGF) and its individual subunitssuch as the 2.5S beta subunit, ion channels, membrane transporters,proteostasis factors, proteins involved in cellular signaling,translation- and transcription related proteins, nucleotide bindingproteins, protein binding proteins, lipid binding proteins,glycosaminoglycans (GAGs) and GAG-binding proteins, metabolic proteins,cellular stress regulating proteins, inflammation and immune systemregulating proteins, mitochondrial proteins, and heat shock proteins,etc. In a preferred embodiment, the therapeutic agent may be aCRISPR-associated (Cas) polypeptide (such as Cas9 (as a non-limitingexample the accession number Q99ZW2)) with intact nuclease activitywhich is associated with (i.e. carries with it) an RNA strand thatenables the Cas polypeptide to carry out its nuclease activity in atarget cell once delivered by the EV. Alternatively, in anotherpreferred embodiment, the Cas polypeptide may be catalytically inactive,to enable targeted genetic engineering. Yet another alternative may beany other type of CRISPR effector such as the single RNA-guidedendonuclease Cpf1 (from species such as Acidaminococcus orLachnospiraceae) (as a non-limiting example the accession number U2UMQ6and A0Q7Q2). Additional preferred embodiments include therapeuticproteins selected from the group comprising enzymes for lysosomalstorage disorders, for instance glucocerebrosidases such asimiglucerase, alpha-galactosidase, alpha-L-iduronidase,iduronate-2-sulfatase and idursulfase, arylsulfatase, galsulfase,acid-alpha glucosidase, sphingomyelinase, galactocerebrosidase,galactosylceramidase, glucosylceramidase (as a non-limiting example theaccession number P04062) ceramidase, alpha-N-acetylgalactosaminidase,beta-galactosidase, lysosomal acid lipase, acid sphingomyelinase, NPC1(as a non-limiting example the accession number 015118), NPC2 (as anon-limiting example the accession number P61916), heparan sulfamidase,N-acetylglucosaminidase, heparan-α-glucosaminide-N-acetyltransferase,N-acetylglucosamine 6-sulfatase, galactose-6-sulfate sulfatase,galactose-6-sulfate sulfatase, hyaluronidase, alpha-N-acetylneuraminidase, GlcNAc phosphotransferase, mucolipin1, palmitoyl-proteinthioesterase, tripeptidyl peptidase I, palmitoyl-protein thioesterase 1,tripeptidyl peptidase 1, battenin, linclin, alpha-D-mannosidase,beta-mannosidase, aspartylglucosaminidase, alpha-L-fucosidase,cystinosin, cathepsin K, sialin, LAMP2, and hexoaminidase. In otherpreferred embodiments, the therapeutic protein may be e.g. anintracellular protein that modifies inflammatory responses, for instanceepigenetic proteins such as methylases and bromodomains, or anintracellular protein that modifies muscle function, e.g. transcriptionfactors such as MyoD (as a non-limiting example the accession numberP15172) or Myf5, proteins regulating muscle contractility e.g. myosin,actin, calcium/binding proteins such as troponin, or structural proteinssuch as dystrophin (as a non-limiting example the accession numberP11532), mini dystrophin (as a non-limiting example the accession numberP15172), utrophin, titin, nebulin, dystrophin-associated proteins suchas dystrobrevin, syntrophin, syncoilin, desmin, sarcoglycan,dystroglycan, sarcospan, agrin, and/or fukutin. Importantly, all of theabove-mentioned therapeutic proteins may be engineered to contain an Fcdomain, in order to enable binding to the Fc binding polypeptide presenton the EVs. Another non-limiting example is the fusion of an Fc domainonto the enzyme NPC1 for subsequent delivery into a target cell. Yetanother non-limiting example which may be utilized to improveintracellular bioactivity of EV-delivered Fc containing proteins (forinstance Fc-Cas9 or antibodies) is to fuse an Fc domain to an endosomalescape peptide or protein, such as HA2, cell-penetrating peptides (CPPs)such as the TAT peptide, transportan, peneratin, poly-lysine, or gp41,cholera toxin, Shiga toxin, saporin, diphtheria toxin peptides, etc.Displaying such endosomal escape domains on the surface of an EV mayenhance both internalization into target cells and subsequent endosomalescape.

An advantageous non-limiting example of how an Fc domain can be fusedonto a protein of interest is the fusion of an Fc domain onto Cas9,Cpf1, non-cleaving Cas variants, or any other type of gene editingprotein or ribonucleoprotein (RNP) for EV-mediated delivery into atarget cell. In a preferred embodiment, an Fc domain is fused eitherN-terminally or C-terminally to Cas9, which has been pre-loaded in vitroor ex vivo with the single guide RNA (sgRNA) strand (Cas pre-loaded withRNA forms a so called ribonucleoprotein (RNP) complex). The resulting Fcdomain-containing RNP complex thus formed is then allowed to be bound bythe Fc binding polypeptides of a suitable EV to attached them to the EVsurface, followed by delivery into target cells. Creation of the RNPcomplex can be achieved in different ways and with different RNAcomponents, such as conventional single guide RNA, a synthetic guide RNAcomprising both the crRNA and the tracrRNA optionally combined with ahairpin loop, crRNA, tracrRNA, and various combinations thereof. Repairtemplates for homology-directed recombination or non-homologousend-joining or any other repair or replacement mechanism may also beincluded in a pre-formed RNP which can then be attached to EVs using theFc domain—Fc binding polypeptide linkage.

The terms “antibody” and “mAb” and “Ab” as described herein is to beunderstood to include both antibodies in their entirety (i.e. wholeantibodies) and any derivatives thereof with antigen-binding properties.Conventionally, an antibody refers to a glycoprotein comprising at leasttwo heavy (H) chains and two light (L) chains inter-connected bydisulfide bonds, or an antigen binding-portion thereof. Each heavy chainis comprised of a heavy chain variable region (abbreviated herein asV_(H)) and a heavy chain constant region. Each light chain is comprisedof a light chain variable region (abbreviated herein as V_(L)) and alight chain constant region. The variable regions of the heavy and lightchains contain a binding domain that interacts with an antigen. TheV_(H) and V_(L) regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Importantly, for the purposes of the present invention anantibody of interest preferably has an Fc domain or a derivative thereofto which the Fc binding polypeptides of the present invention can bind,in order to enable coating of the EV surface. Antibodies of use in theinvention may be monoclonal antibodies (mAbs) or polyclonal antibodies,preferably mAbs. Antibodies of particular utility in the invention maybe chimeric antibodies, CDR-grafted antibodies, nanobodies, human orhumanised antibodies or any derivative thereof as long as it can bebound by the Fc binding polypeptide, which are typically comprised inthe fusion proteins as per the present invention. The production ofantibodies is outside of the scope of the present invention buttypically both monoclonal and polyclonal antibodies are raisedexperimental non-human mammals such as goat, rabbit, llama, camelids,rat or mouse, but suitable antibodies may also be the result of otherproduction methodologies, e.g. the standard somatic cell hybridizationtechnique of Kohler and Milstein. Hybridoma production in e.g. the mouseis a very well-established procedure and can be achieved usingtechniques well known in the art. An antibody of use in the inventionmay be a human antibody, humanized antibody, and/or any type of chimericantibody. The term “human antibody”, as used herein, is intended toinclude antibodies having variable regions in which both the frameworkand CDR regions are derived from human germline immunoglobulinsequences. The human antibodies of use in the invention may includeamino acid residues not encoded by human germline immunoglobulinsequences (e.g., mutations introduced by random or site-specificmutagenesis in vitro or by somatic mutation in vivo). The term “antibodyderivatives” refers to any modified form of an antibody, e.g. anantibody having an amino acid sequence that is modified in any way, or aconjugate of the antibody and another agent or antibody, bispecificantibodies, multispecific antibodies, antibody domains, etc. The term“humanized antibody” refers to antibodies in which CDR sequences derivedfrom another mammalian species, such as a mouse, camelid, llama, etc.,have been grafted onto human framework sequences. Additional frameworkregion modifications may be made within the human framework sequences.Antibodies in accordance with the present invention may include allisotypes and subtypes such as IgG (for instance IgG1, IgG2, IgG3, IgG4,IgG2a, IgG2d, and IgG2c), IgA, IgM, IgM, IgD, etc., and monomers,dimers, and oligomers thereof. Further, antibodies as per the presentinvention may have several functions when displayed on EVs: (1)antibodies may target specific cell types, tissues, and/or organs, (2)diagnostic and/or therapeutic antibodies that interact with a targetantigen of interest can be efficiently delivered to tissues of interestusing EVs, (3) multiplexed antibodies on the surface of EVs may besignificantly better at binding target antigens, (4) antibody-drugconjugates (ADCs) may be multiplexed on EVs to enhance the potency ofe.g. a radionuclide attached to the antibody, (5) antibodies bound by Fcbinding polypeptide may have higher affinity for the target because ofthe binding between Fc binding polypeptide and the antibody Fc domain,(6) antibodies coated onto EVs as per the present invention may bedelivered intracellularly for diagnostic and/or therapeutic and/orimaging and/or research purposes. Importantly, antibodies and other Fccontaining proteins as per the present invention are advantageouslylabelled with some form of detection moiety. The presence of a detectionmoiety, such as a fluorophore, a radioactive substance, a PET tracer,MRI agents, or any other type of detection moiety, is highlyadvantageous for tracking the labelled Fc containing protein and hencethe EVs, either in vivo, in vitro and/or ex vivo. Accordingly, tracking,tracing and detection can thus be achieved using a wide variety ofdifferent methods, such as PET scans, confocal microscopy andfluorescence microscopy, electron microscopy, magnetic resonance imaging(MRI), X-ray scans, CAT scans, flow cytometry, nanoparticle trackinganalysis, etc.

The terms “Fc containing protein” and “protein comprising an Fc domain”and “Fc domain-containing protein” and “Fc domain containing protein”and “Fc domain protein” and similar terms are used interchangeablyherein and shall be understood to relate to any protein, polypeptide, orpeptide (i.e. any molecule comprising a sequence of amino acids) whichcomprises at least one Fc domain, either naturally or as a result ofengineering of the protein in question to introduce an Fc domain. Fcstands for “fragment crystallizable”, which is the name of the tailregions of antibodies. Fc domains can however also be created and usedon other proteins, not only antibodies. Non-limiting examples of such Fcdomain-containing proteins include antibodies and antibody derivatives,Fc-modified decoy receptors and/or signal transducers such asinterleukin decoy receptors for IL1, IL2, IL3, IL4, IL5, IL6 (such asthe signal transducer gp130 (as a non-limiting example the accessionnumber P40189)), IL7, IL8, IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL17(such as IL17R, with as a non-limiting example the accession numberQ96F46), IL23 (such as IL23R, as a non-limiting example the accessionnumber Q5VWK5), etc., Fc domain-containing bi- and multispecificbinders, any type of Fc domain-containing receptors or ligands, Fcdomain-modified enzymes for e.g. enzyme replacement therapy or geneediting, nucleases such as Cas and Cas9 onto which an Fc domain has beengrafted, tumor suppressors fused to Fc domains, etc. Suitable Fc domainsthat may be fused with a protein of interest natively lacking an Fcdomain include the following non-limiting examples: human IGHM (as anon-limiting example the accession number P01871), human IGHA1 (as anon-limiting example the accession number P01876), human IGHA2 (as anon-limiting example the accession number P01877), human IGKC (as anon-limiting example the accession number P01834), human IGHG1 (as anon-limiting example the accession number P01857), human IGHG2 (as anon-limiting example the accession number P01859), human IGHG3 (as anon-limiting example the accession number P01860), human IGHG4 (as anon-limiting example the accession number P01861), human IGHD (as anon-limiting example the accession number P01880), human IGHE (as anon-limiting example the accession number P01854), and any domains,derivatives, or combinations thereof. In essence, any protein ofinterest may be modified to incorporate an Fc domain. Non-limitingexamples of proteins onto which an Fc domain can be introduced includefor instance tumor suppressors, viral or bacterial inhibitors, cellcomponent proteins, DNA and/or RNA binding proteins, DNA repairinhibitors, nucleases, proteinases, integrases, transcription factors,growth factors, apoptosis inhibitors and inducers, toxins (for instancepseudomonas exotoxins), structural proteins, neurotrophic factors suchas NT3/4, brain-derived neurotrophic factor (BDNF) and nerve growthfactor (NGF) and its individual subunits such as the 2.5S beta subunit,ion channels, membrane transporters, proteostasis factors, proteinsinvolved in cellular signaling, translation- and transcription relatedproteins, nucleotide binding proteins, protein binding proteins, lipidbinding proteins, glycosaminoglycans (GAGs) and GAG-binding proteins,metabolic proteins, cellular stress regulating proteins, inflammationand immune system regulating proteins, mitochondrial proteins, and heatshock proteins, etc. The above list of proteins of interest is notexhaustive and other proteins may also be relevant, as long as theprotein either comprises an Fc domain or as long as it is possible toengineer the protein in question to comprise an Fc domain. Onenon-limiting example of such engineering of a protein to introduce an Fcdomain includes adding an Fc domain to a decoy receptor, e.g. adding anFc domain onto the Cas9 enzyme for bioactive delivery into target cellsto enable gene editing. Another non-limiting example of such engineeringof a protein to introduce an Fc domain includes adding an Fc domain ontoan enzyme for enzyme replacement therapy (for instance Fcdomain-glucocerebrosidase or Fc domain-α-galactosidase or Fcdomain-NPC1). A well-known example of a commercially available Fcdomain-modified protein is etanercept, which is a biopharmaceutical forthe treatment of various autoimmune disease, comprising the Fc domain ofIgG fused onto TNF receptor 2. Thus, as is clear from the above, Fcdomain-containing proteins as per the present invention may beessentially any protein of interest that contains an Fc domain, eithernaturally or as a result of introduction thereof. As above-mentioned inconnection with antibodies, all Fc domain containing proteins may bemodified to incorporate a detection moiety, in order to enable detectionand tracing of the molecule in question and/or of the EV in question.

The terms “Fc binding polypeptide” and “Fc binding protein” and “Fcbinder” and “Fc-binding protein” and “binder” are used interchangeablyherein and shall be understood to relate to any protein, polypeptide, orpeptide (i.e. any molecule comprising a sequence of amino acids) whichcan bind an Fc domain of any protein of interest. Typically, the Fcbinding polypeptides of the present invention are derived from varioussources that are either human or non-human (e.g. mammal sources,bacteria, etc.), they have high affinity for Fc domains of variousantibody isotypes, subtypes, and species (for instance IgG (and morespecifically IgG1, IgG2, IgG3, IgG4, IgG2a, IgG2d, and/or IgG2c), IgA,IgM, IgM, IgD, etc.), and they can be fused to EV proteins. Non-limitingexamples of Fc binding polypeptides in accordance with the presentinvention include, in addition to other Fc binding polypeptidesmentioned through the present application, Protein A (as a non-limitingexample the SEQ ID NO 77), Protein G (as a non-limiting example the SEQID NO 78), Protein A/G (as a non-limiting example the SEQ ID NO 72), Zdomain (as a non-limiting example the SEQ ID NO 73), ZZ domain (twooperably linked copies of as a non-limiting example the SEQ ID NO 73,i.e. as a non-limiting example the SEQ ID NO 74), human FCGRI (as anon-limiting example the SEQ ID NO 31), human FCGRIIA (as a non-limitingexample the SEQ ID NO 33), human FCGRIIB (as a non-limiting example theaccession number 31994), human FCGRIIC (as a non-limiting example theaccession number 31995), human FCGRIIIA (as a non-limiting example theaccession number P08637), human FCGR3B (as a non-limiting example theaccession number 075015), human FCAMR (as a non-limiting example the SEQID NO 28), human FCERA, human FCAR, mouse FCGRI (as a non-limitingexample the SEQ ID NO 79), mouse FCGRIIB (as a non-limiting example theSEQ ID NO 80), mouse FCGRIII (as a non-limiting example the SEQ ID NO81), mouse FCGRIV (as a non-limiting example the SEQ ID NO 82), mouseFCGRn (as a non-limiting example the SEQ ID NO 83), and variouscombinations, derivatives, or alternatives thereof.

The Fc containing proteins are often described herein as being “attachedto” an EV and/or to an Fc binding polypeptide. Alternatively, EVs aresometimes referred to as being “coated by” Fc containing proteins, or ashaving “bound to their surface” or “attached to their surface” the Fccontaining proteins. These terms shall be understood in the context ofthe conventional interaction between an Fc binding polypeptide and an Fcdomain, that is that the two polypeptides are interacting with eachother in a way that results in a chemical bond (typically a non-covalentbond) forming between the Fc binder and the Fc domain. Thus, thisnormally means that the EV which comprises the Fc binding polypeptidetherefore has attached to it, by virtue of the chemical bond, the Fcdomain of the Fc containing protein. As will be understood by theskilled person, an EV may consequently have a plurality of such Fccontaining proteins bound (attached) to it, resulting in a form ofcoating when the binding is taking place on the EV surface.

The terms “EV protein” and “EV polypeptide” and “exosomal polypeptide”and “exosomal protein” are used interchangeably herein and shall beunderstood to relate to any polypeptide that can be utilized totransport a polypeptide construct (which typically comprises, inaddition to the EV protein, at least one Fc binding polypeptide andoptionally additional polypeptides and/or linkers) to a suitablevesicular structure, i.e. to a suitable EV. More specifically, theseterms shall be understood as comprising any polypeptide that enablestransporting, trafficking or shuttling of a fusion protein construct toa vesicular structure, such as an EV. Examples of such exosomalpolypeptides are for instance CD9 (as a non-limiting example the SEQ IDNO 1), CD53 (as a non-limiting example the SEQ ID NO 2), CD63 (as anon-limiting example the SEQ ID NO 3), CD81 (as a non-limiting examplethe SEQ ID NO 4), CD54 (as a non-limiting example the SEQ ID NO 5), CD50(as a non-limiting example the SEQ ID NO 6), FLOT1 (as a non-limitingexample the SEQ ID NO 7), FLOT2 (as a non-limiting example the SEQ ID NO8), CD49d (as a non-limiting example the SEQ ID NO 9), CD71 (also knownas the transferrin receptor) (as a non-limiting example the SEQ ID NO10) and its endosomal sorting domain, i.e. the transferrin receptorendosomal sorting domain (as a non-limiting example the SEQ ID NO 23),CD133 (as a non-limiting example the SEQ ID NO 11), CD138 (syndecan-1)(as a non-limiting example the SEQ ID NO 12), CD235a (as a non-limitingexample the SEQ ID NO 13), ALIX (as a non-limiting example the SEQ ID NO14), Syntenin-1 (as a non-limiting example the SEQ ID NO 15), Syntenin-2(as a non-limiting example the SEQ ID NO 16), Lamp2b (as a non-limitingexample the SEQ ID NO 17), syndecan-2 (as a non-limiting example the SEQID NO 20), syndecan-3 (as a non-limiting example the SEQ ID NO 21),syndecan-4 (as a non-limiting example the SEQ ID NO 22), TSPAN8,TSPAN14, CD37, CD82, CD151, CD231, CD102, NOTCH1, NOTCH2, NOTCH3,NOTCH4, DLL1, DLL4, JAG1, JAG2, CD49d/ITGA4, ITGB5, ITGB6, ITGB7, CD11a,CD11b, CD11c, CD18/ITGB2, CD41, CD49b, CD49c, CD49e, CD51, CD61, CD104,Fc receptors, interleukin receptors, immunoglobulins, MHC-I or MHC-IIcomponents, CD2, CD3 epsilon, CD3 zeta, CD13, CD18, CD19 (as anon-limiting example the SEQ ID NO 26), CD30 (as a non-limiting examplethe SEQ ID NO 27), TSG101, CD34, CD36, CD40, CD40L, CD44, CD45, CD45RA,CD47, CD86, CD110, CD111, CD115, CD117, CD125, CD135, CD184, CD200,CD279, CD273, CD274, CD362, COL6A1, AGRN, EGFR, GAPDH, GLUR2, GLUR3,HLA-DM, HSPG2, L1CAM, LAMB1, LAMC1, LFA-1, LGALS3BP, Mac-1 alpha, Mac-1beta, MFGE8, SLIT2, STX3, TCRA, TCRB, TCRD, TCRG, VTI1A, VTI1B, otherexosomal polypeptides, and any combinations thereof, but numerous otherpolypeptides capable of transporting a polypeptide construct to an EVare comprised within the scope of the present invention. Typically, inmany embodiments of the present invention, at least one exosomalpolypeptide is fused to at least one Fc binding polypeptide, in order toform a fusion protein present in an EV. Such fusion proteins may alsocomprise various other components to optimize their function(s),including linkers, transmembrane domains, cytosolic domains,multimerization domains, etc.

The terms “source cell” or “EV source cell” or “parental cell” or “cellsource” or “EV-producing cell” or any other similar terminology shall beunderstood to relate to any type of cell that is capable of producingEVs under suitable conditions, for instance in suspension culture or inadherent culture or any in other type of culturing system. Source cellsas per the present invention may also include cells producing exosomesin vivo. The source cells per the present invention may be select from awide range of cells and cell lines, for instance mesenchymal stem orstromal cells or fibroblasts (obtainable from e.g. bone marrow, adiposetissue, Wharton's jelly, perinatal tissue, tooth buds, umbilical cordblood, skin tissue, etc.), amnion cells and more specifically amnionepithelial cells optionally expressing various early markers, myeloidsuppressor cells, M2 polarized macrophages, adipocytes, endothelialcells, fibroblasts, etc. Cell lines of particular interest include humanumbilical cord endothelial cells (HUVECs), human embryonic kidney (HEK)cells, endothelial cell lines such as microvascular or lymphaticendothelial cells, chondrocytes, MSCs of different origin, airway oralveolar epithelial cells, fibroblasts, endothelial cells, etc. Also,immune cells such as B cells, T cells, NK cells, macrophages, monocytes,dendritic cells (DCs) are also within the scope of the presentinvention, and essentially any type of cell which is capable ofproducing EVs is also encompassed herein. Generally, EVs may be derivedfrom essentially any cell source, be it a primary cell source or animmortalized cell line. The EV source cells may be any embryonic, fetal,and adult somatic stem cell types, including induced pluripotent stemcells (iPSCs) and other stem cells derived by any method. When treatingneurological diseases, one may contemplate to utilize as source cellse.g. primary neurons, astrocytes, oligodendrocytes, microglia, andneural progenitor cells. The source cell may be either allogeneic,autologous, or even xenogeneic in nature to the patient to be treated,i.e. the cells may be from the patient himself or from an unrelated,matched or unmatched donor. In certain contexts, allogeneic cells may bepreferable from a medical standpoint, as they could provideimmuno-modulatory effects that may not be obtainable from autologouscells of a patient suffering from a certain indication. For instance, inthe context of treating systemic, peripheral and/or neurologicalinflammation, allogeneic MSCs may be preferable as EVs obtainable fromsuch cells may enable immuno-modulation via e.g. macrophage and/orneutrophil phenotypic switching (from pro-inflammatory M1 or N1phenotypes to anti-inflammatory M2 or N2 phenotypes, respectively).

In a first aspect, the present invention relates to the use of EVscomprising at least one Fc binding polypeptide as a research tool.Non-limiting uses of relevance include the use of the Fc binding EVs ina detection kit, as a biological RM, as a control and/or a standard inflow cytometry, imaging flow cytometry, nanoparticle tracking analysis,nanoimaging, confocal microscopy, fluorescence microscopy, dynamic lightscattering, electron microscopy, luminescence-based detection,fluorescence correlation spectroscopy, fluorescence resonance energytransfer (FRET)-based techniques, positive or negative selectiontechniques based on magnetic, fluorescent, and/or buoyancy-basedlabelling, positive or negative selection techniques based on particledensity, charge or size, or in essentially any other technique or methodwherein an EV with the ability to bind to Fc domain containing proteins(such as antibodies) can be useful. In advantageous embodiments, the atleast one Fc binding polypeptide which is comprised in the EV forms partof a fusion protein together with at least one exosomal polypeptide, andoptionally other polypeptide domains and/or regions.

The EVs as per the present invention are highly advantageous for variousresearch, diagnostic, imaging, and therapeutic uses in particular whenthe Fc binding polypeptide of the EV binds to a protein comprising an Fcdomain. The binding between the Fc binding polypeptide of the EVs and anFc containing protein results in the formation of a nano-sized EV-Fcprotein complex, with extraordinary utility in e.g. flow cytometry, as atransport vehicle for Fc containing proteins such as antibodies, innanoparticle tracking analysis, in dynamic light scattering, in electronmicroscopy, and/or in various other types techniques. In preferredembodiments, the use of the EVs as per the present invention includesattaching an antibody to the at least one Fc binding polypeptide, and ineven more preferred embodiments the antibody is labelled with some formof detection moiety. Suitable detection moieties and/or agents which maybe attached the antibodies (or to any other Fc containing protein) arefluorophores, radioactive labels, PET ligands, MRI agents,nanoparticles, inorganic and/or compounds, enzymes, or any othersuitable detection moiety adapted to the method in which the EVs areused.

In one aspect, the present invention provides for a vesicular referencematerial (RM) based on the EVs disclosed herein. The term RM can meanany material that can be used for the purpose of standardization,positive control, calibration control, and/or compensation control orany other type of reference control, in any kind of assay, method orapplication. In the context of RMs for EV studies and analyses, theoptimal RM would be a biologically derived vesicle, which is why the Fcbinding polypeptide-containing EVs of the present are optimally suitedfor this purpose. Thus, in one embodiment, the present invention relatesto an RM comprising a population of EVs as described herein, i.e. EVscomprising an Fc binding polypeptide, which optionally is comprised in afusion protein with at least one EV protein. In an advantageousembodiment, the Fc binding polypeptides bind to an Fc containing protein(such as an antibody), which adds an additional layer of information andfunctionality to the RM. The RM may be present in any physical form,such as in a liquid, in a powder, in an aerosol, as a freeze-driedmaterial, etc. As above-mentioned, biologically relevant RMs are highlysought after not only within the EV field but also more broadly acrossthe sciences and the engineered EVs of the present invention representan almost perfect RM for many applications from flow cytometry andnanoparticle tracking analysis to fluorescence microscopy and electronmicroscopy.

In one embodiment, the at least one Fc binding polypeptide comprised inthe EV forms part of a fusion protein together with at least oneexosomal polypeptide. The fusion with at least one EV protein normallyenhances the trafficking to EVs and subsequently the incorporationand/or display of the Fc binding polypeptide. The Fc binding polypeptidepresent in and/or on the EV is clearly intended to bind to, and therebyattach to the EV, proteins containing Fc domains, such as antibodiesand/or proteins onto which an Fc domain has been fused. The bindingnaturally takes place between the Fc binding polypeptide of the EV andthe Fc domain of the protein of interest, typically via a conventionalnon-covalent interaction.

As above-mentioned, in a preferred embodiment the biological RM hasattached to it an Fc containing protein in the form of an antibody. Infurther advantageous embodiments, Fc containing protein has attached toit at least one fluorophore, at least one radioactive label, at leastone PET ligand, at least one MRI agent, or any other detection moiety,in order to enable detection, tracing and imaging of the RM in vitro, invivo, ex vivo, or in any research setting. The research settings inwhich the biological RM may be used include the following non-limitingexamples: flow cytometry, imaging flow cytometry, nanoparticle trackinganalysis, nanoimaging, confocal microscopy, fluorescence microscopy,dynamic light scattering, electron microscopy, luminescence-baseddetection, fluorescence correlation spectroscopy, fluorescence resonanceenergy transfer (FRET)-based techniques, positive or negative selectiontechniques based on magnetic, fluorescent, and/or buoyancy-basedlabelling, positive or negative selection techniques based on particledensity, charge or size, or any combination thereof.

In a further aspect, the present invention relates to an in vitro, invivo and/or ex vivo method for delivery of at least one type of Fccontaining protein into a target cell, comprising (i) providing an EVcomprising at least one Fc binding polypeptide, (ii) allowing the Fcbinding polypeptide to bind the Fc containing protein, and (iii)contacting a target cell with the EV having attached to its Fc bindingpolypeptide the Fc containing protein. In one embodiment, the at leastone Fc binding polypeptide may form part of a fusion protein togetherwith at least one exosomal polypeptide, and optionally with variousother polypeptides (such as linkers, multimerization domains, cytokinereceptors and signal transducers, etc.). In one advantageous embodiment,the Fc containing protein is an antibody. In another advantageousembodiment, the Fc containing protein is attached to at least onefluorophore, at least one radioactive label, at least one PET ligand, orany other detection moiety.

In yet another aspect, the present invention relates to a detection kitcomprising an EV, wherein the EV comprises at least one Fc bindingpolypeptide (fused to at least one exosomal polypeptide), wherein the Fcbinding polypeptide is optionally bound to at least one an Fc containingprotein. Antibodies are clearly particularly suited for detection ofvarious target antigens and with the aid of the EVs as per the presentinvention not only extracellular but also intracellular targets can beprobed. To enable detection, the Fc containing protein (e.g. at leastone type of antibody) may be labelled with a detection moiety. Thenature of the detection moiety is completely dependent on the detectionmethod being applied but suitable detection moieties may be select fromfor instance the following non-limiting examples: a fluorophore, aradioactive label, a PET ligand, an MRI agent, an enzyme for enzymaticdetection, a colorimetric detection moiety, a bioluminescence and/orchemiluminescence detection moiety, inorganic or organic compounds,metals, quantum dots, nanoparticles (for instance gold nanoparticles),or any other detection moiety dependent on the detection method oneintends to applied.

As above-mentioned, the present invention allows for detection bothinside and outside target cells, and also in the cell membrane, meaningthat extracellular, membrane-associated and/or intracellularbiomolecules can be probed using the EV-based detection kit of thepresent invention. Furthermore, the detection of a particularbiomolecule may take place in vitro, in vivo and/or ex vivo. The sample(such as an animal, tissue, organ, cell, subcellular organelle, bodyfluid, etc.) may be visualize and/or imaged either alive or dead, e.g.frozen, preserved, fixed or conserved for e.g. immuno-histochemistry, oron slides and grids for microscopy and electron microscopy.

The detection kit as per the present invention may be utilized in flowcytometry, nanoparticle tracking analysis, nanoimaging, confocalmicroscopy, fluorescence microscopy, dynamic light scattering, electronmicroscopy, luminescence-based detection, fluorescence correlationspectroscopy, fluorescence resonance energy transfer (FRET)-basedtechniques, positive or negative selection techniques based on magnetic,fluorescent, and/or buoyancy-based labelling, positive or negativeselection techniques based on density, charge or size, or anycombination of such methods. Typically, the detection kit would bedesigned or formulated to enable the bond between the Fc bindingpolypeptide-containing EVs and the Fc containing protein to remainintact under various conditions, and occasionally to release the Fccontaining proteins under other conditions. The complex between the EVsand the Fc containing proteins may be present in various physical andchemical states, e.g. in solution, in a gel, in an emulsion, in asuitable buffer, in lyophilized form, in nebulized form, in particulateform, in a hydrogel, in a liposomal formulation, in a polymerformulation, etc.

In a further aspect, the present invention relates to a kit of partscomprising (i) an EV comprising at least one Fc binding polypeptide, and(ii) at least one Fc containing protein. The at least one Fc bindingpolypeptide may be comprised in a fusion protein together with at leastone exosomal polypeptide and optionally other polypeptides and domainsthereof. The components (i) and (ii) may be present e.g. in differentcontainers, or in the same container, depending on e.g. how the kit isto be used, stored, sold, and shipped. The components (i) and (ii) maybe mixed immediately prior to use or they may be pre-mixed and shippedas a nanoparticle complex between the EVs and the Fc containing protein.

In yet another aspect, the present invention pertains to a nanoparticlecomplex between (i) an EV comprising at least one Fc binding polypeptideand (ii) an Fc containing protein. The at least one Fc bindingpolypeptide may form part of a fusion protein together with at least oneexosomal polypeptide, in order to increase its incorporation into the EVand/or the display of the Fc binding polypeptide on the surface of anEV. The nanoparticle complex formed between the engineered EV and the Fccontaining protein can be schematically described as a nanoparticlecoated with the Fc containing protein (as an example, multiple copies ofan antibody). Antibodies constitute a particularly preferred embodimentof the present invention, as they exhibit high target specificity, areeasy to develop, can be modified to incorporate detection moieties and,with the aid of the EVs of the present invention, can be delivered intotarget cells. The nanoparticle complexes of the present invention havebroad utility across research, diagnostics, imaging, analytics andtherapy, and may be used as biological RM, as a research tool, as adiagnostic tool, an imaging tool, a detection kit, and/or for in vitro,in vivo and/or ex vivo delivery of different types of Fc containingproteins.

In one embodiment, the EVs themselves comprise a detection moiety, whichmay optionally be combined with a detection moiety attached to the Fccontaining protein (such as a fluorescently labelled antibody). SinceEVs are nano-sized vesicles various types of detection moieties may beused, for instance fluorophores, dyes, fluorescent proteins such asgreen fluorescent protein, red fluorescent protein, yellow fluorescentprotein, and any fluorescent protein; firefly or renilla luciferase,NanoLuciferase and other types of enzymes producing bioluminescencesignals; radioactive agents; PET and MRI agents and tracers;nanoparticles, inorganic compounds, metals and metal ions, eithersolubilized or in various complexes, etc. An advantageous embodiment isEVs comprising GFP and/or luciferase/NanoLuciferase, combined with e.g.fluorescently labelled antibodies (or any other Fc containing protein)attached to the Fc binding polypeptides of the EVs. Essentially, addingany other type of detection moiety to the EV as such provides fordetection through more than one means which may be highly useful in e.g.microscopy, flow cytometry, nanoparticle tracking analysis, dynamiclight scattering, and in various other techniques.

The Fc binding polypeptides of the present invention may be of non-humanorigin or of human origin. The Fc binders may be obtained e.g. frombacteria, viruses, or non-human mammals. In another embodiment, the Fcbinders are of human or mammal origin. In preferred embodiments, the atleast one Fc binding polypeptide may be selected from the groupcomprising Protein A (as a non-limiting example the SEQ ID NO 77),Protein G (as a non-limiting example the SEQ ID NO 78), Protein A/G (asa non-limiting example the SEQ ID NO 72), Z domain (as a non-limitingexample the SEQ ID NO 73), ZZ domain (as a non-limiting example the SEQID NO 74), Protein L (as a non-limiting example the pdb id no 1HEZ),Protein LG, human FCGRI (as a non-limiting example the SEQ ID NO 31),human FCGR2A (as a non-limiting example the accession number P12318),human FCGR2B (as a non-limiting example the accession number P31994),human FCGR2C (as a non-limiting example the accession number P31994),human FCGR3A (as a non-limiting example the accession number P08637),human FCGR3B (as a non-limiting example the accession number O75015),human FCGRB (as a non-limiting example the accession number Q92637) (asa non-limiting example the SEQ ID NO 32), human FCAMR (as a non-limitingexample the SEQ ID NO 28), human FCERA (as a non-limiting example theSEQ ID NO 30), human FCAR (as a non-limiting example the SEQ ID NO 29),mouse FCGRI (as a non-limiting example the SEQ ID NO 79), mouse FCGRIIB(as a non-limiting example the SEQ ID NO 80), mouse FCGRIII (as anon-limiting example the SEQ ID NO 81), mouse FCGRIV (as a non-limitingexample the SEQ ID NO 82), mouse FCGRn (as a non-limiting example theSEQ ID NO 83), and any combination of any of the above Fc bindingpolypeptides. Other suitable Fc binding polypeptides, which have beenobtained from e.g. phage display screening and via bioinformatics,include the Fc binding peptides SPH (as a non-limiting example the SEQID NO 57), SPA (as a non-limiting example the SEQ ID NO 58), SPG2 (as anon-limiting example the SEQ ID NO 59), SpA mimic 1 (as a non-limitingexample the SEQ ID NO 60), SpA mimic 2 (as a non-limiting example theSEQ ID NO 61), SpA mimic 3 (as a non-limiting example the SEQ ID NO 62),SpA mimic 4 (as a non-limiting example the SEQ ID NO 63), SpA mimic 5(as a non-limiting example the SEQ ID NO 64), SpA mimic 6 (as anon-limiting example the SEQ ID NO 65), SpA mimic 7 (as a non-limitingexample the SEQ ID NO 66), SpA mimic 8 (as a non-limiting example theSEQ ID NO 69), SpA mimic 9 (as a non-limiting example the SEQ ID NO 70),SpA mimic 10 (as a non-limiting example the SEQ ID NO 71), Fcγ mimic 1(as a non-limiting example the SEQ ID NO 67), and Fcγ mimic 2 (as anon-limiting example the SEQ ID NO 68), and any combination orderivative thereof. The selection of the most suitable Fc bindingpolypeptide for a particular construct depends heavily on the desiredbinding characteristics, the affinity, the orientation of the Fc bindingpolypeptide when fused to an exosomal polypeptide, and various otherfactors.

Protein A/G is a recombinant genetically engineered protein comprised of7 Fc-binding domains EDABC-C1C3, with the Protein A part being obtainedfrom Staphylococcus aureus segments E, D, A, B and C, and the Protein Gpart from Streptococcus segments C1 and C3. Advantageously, Protein A/G(as a non-limiting example the SEQ ID NO 72) has a broader bindingcapacity than either Protein A (as a non-limiting example the SEQ ID NO77) or Protein G (as a non-limiting example the SEQ ID NO 78) alone andit has a broad binding affinity for antibodies from various species.Protein A/G binds to various human, mouse and rat IgG subclasses such asthe human IgG1, IgG2, IgG3, IgG4; mouse IgG2a, IgG2b, IgG3 and ratIgG2a, IgG2c. In addition, Protein A/G binds to total IgG from cow,goat, sheep, horse, rabbit, guinea pig, pig, dog and cat. Protein A/Ghas been engineered to remove the cell wall-binding region, the cellmembrane-binding region and albumin-binding region to enable strongbinding to the Fc domain of a protein of interest, such as an antibody.Thus, in advantageous embodiments as per the present invention, the Fcbinder comprises more than one Fc binding region, as is the case withProtein A, Protein G, and Protein A/G, in order to enable binding a widerange of antibodies. In an alternative embodiment, the Fc binder may bemultiplied in order to enable binding to more than one copy of anantibody of interest. For instance, the short Z domain Fc binder may beincluded in the fusion protein in more than one copy, through anoperational linkage allowing for binding to more than one Fc domain.This way it is possible to multiplex antibodies and other Fcdomain-containing proteins not only between separate fusion proteins butalso within one single fusion protein, which thus may bind more than oneantibody. For instance, when Fc binding polypeptides are introduced intoEV proteins belonging to the tetraspanin family (such as CD63) it may beadvantageous to insert one Fc binder on one loop and another Fc binder(which can be the same or different) on another loop of the protein. TheFc binder can be placed on inward-facing and/or outward-facing loops,depending on whether the Fc containing protein is meant to be loadedinto the lumen of the EV or onto the surface of the EV. Somenon-limiting examples of fusion proteins as per the present inventioncan be described schematically as follows (the below notation is not tobe construed as illustrating any C and/or N terminal direction, it ismerely meant for illustrative purposes):

-   -   Exosomal polypeptide-Fc binding polypeptide-Fc binding        polypeptide    -   Exosomal polypeptide domain-Fc binding polypeptide-Exosomal        polypeptide domain-Fc binding polypeptide    -   Exosomal polypeptide domain-Fc binding polypeptide A-Exosomal        polypeptide domain-Fc binding polypeptide B

In some embodiments, the fusion proteins comprising the exosomalpolypeptide and the Fc binding polypeptide may also contain additionalpolypeptides, polypeptide domains or sequences. Such additionalpolypeptide domains may exert various functions, for instance suchdomains may (i) contribute to enhancing the EV surface display of thefusion protein, (ii) lead to clustering of the fusion proteins therebyincreasing the avidity of the Fc binding polypeptides, (iii) function aslinkers to optimize the interaction between the exosomal polypeptidesand the Fc binding polypeptide, and/or (iv) improve anchoring in the EVmembrane, as well as various other functions. Two such additionalpolypeptides that may advantageously be included in part or as a wholein the fusion proteins of the preset invention are gp130 (as anon-limiting example the SEQ ID NO 18) and the tumor necrosis factorreceptor 1 (TNFR1) (as a non-limiting example the SEQ ID NO 19). Inparticular, the transmembrane domains of these additional polypeptidesmay be highly useful to optimize the insertion into the EV membrane andthe display of the Fc binding polypeptide. Overall, varioustransmembrane domains may be highly advantageous as additional domainsin the fusion proteins. For instance, when using the exosomal proteinsyntenin it is highly advantageous to insert an additional polypeptidedomain, such the transmembrane domain of TNFR or the transmembranedomain of gp130, for instance between syntenin and the Fc bindingpolypeptide of the fusion protein. Further additional domains mayinclude multimerization domains such as fold-on domains, leucine zipperdomains and/or trimerization domains, in order to increase surfacedisplay and/or avidity. A non-limiting schematic example of this isshown below:

-   -   Syntenin-cytosolicTNRF-Foldon trimerization        domain-transmembraneTNFR domain-Z domain-extracellularTNFR        domain    -   Syndecan-leuzine zipper domain-gp130cytosolic domain-gp130        transmembrane domaine-Fc binding polypeptide-gp130 extracellular        domain

In a further embodiment, the exosomal polypeptide may be selected fromthe group comprising CD9, CD53, CD63, CD81, CD54, CD50, FLOT1, FLOT2,CD49d, CD71, CD133, CD138, CD235a, ALIX, Syntenin-1, Syntenin-2, Lamp2b,TSPAN8, syndecan-1, syndecan-2, syndecan-3, syndecan-4, TSPAN14, CD37,CD82 (as a non-limiting example the SEQ ID NO 24), CD151, CD231, CD102,NOTCH1, NOTCH2, NOTCH3, NOTCH4, DLL1, DLL4, JAG1, JAG2, CD49d/ITGA4,ITGB5, ITGB6, ITGB7, CD11a, CD11b, CD11c, CD18/ITGB2, CD41, CD49b,CD49c, CD49e, CD51, CD61, CD104, Fc receptors, interleukin receptors,immunoglobulins, MHC-I or MHC-II components, CD2, CD3 epsilon, CD3 zeta,CD13, CD18, CD19, CD30, CD34, CD36, CD40, CD40L, CD44, CD45, CD45RA,CD47, CD86, CD110, CD111, CD115, CD117, CD125, CD135, CD184, CD200,CD279, CD273, CD274, CD362, COL6A1, AGRN, EGFR, GAPDH, GLUR2, GLUR3,HLA-DM, HSPG2, L1CAM, LAMB1, LAMC1, LFA-1, LGALS3BP, Mac-1 alpha, Mac-1beta, MFGE8 (as a non-limiting example the SEQ ID NO 25), SLIT2, STX3,TCRA, TCRB, TCRD, TCRG, VTI1A, VTI1B, other exosomal polypeptides, andany combinations thereof.

Particularly advantageous fusion proteins as per the present inventionmay comprise the human exosomal proteins CD63, CD81, CD9, CD71(transferrin receptor), Lamp2, and syntenin, fused to at least one copyof the following Fc binding polypeptides: Z domain, Protein A, ProteinG, Protein A/G, FCGRI, human FCGR2A, human FCGR2B, human FCGR2C, humanFCGR3A, human FCGR3B, human FCGR3C, human FCAMR, human FCAR, and humanFCERA. Specifically, fusion of two Z domains to the EV protein CD63 (asa non-limiting example the SEQ ID NO 3) results in a highly potent Fcbinder which is displayed in high quantities on the surface of EVs, as aconsequence of the small size of the Z domain and the high EV surfaceexpression of CD63. Display of the Fc binder on the outer surface of EVsis naturally preferable in order to enable exogenous binding of Fcdomain-containing proteins of interest. In the case of CD63-ZZ domainfusion proteins, Z domains may be inserted into the first and secondloop of CD63, to enable display on the outer EV surface after anchoringof CD63 into the EV membrane. Furthermore, as above-mentioned, suitableadditional polypeptides and polypeptide domains and linkers may also beincluded in the fusion proteins, which normally comprises an exosomalpolypeptide and an Fc binding polypeptide. Such additional polypeptidesinclude domains from various cytokine receptors such as TNFR1 and TNFR2,the IL6 signal transducer gp130, and various other cytokines andcytokine-related polypeptides. In certain instances, cytokines andcytokine-related polypeptides (such as various cytokine receptors) mayalone be able to transport Fc binding polypeptides to EVs. Linkers ofparticular utility are multiples and/or combinations of small andflexible amino acids such as glycine (G) and serine (S), typicallydenoted GS, for instance 2XGS or 4XGS. His tags for simplifyingpurification and assaying may also be added, both C terminally and Nterminally, and so may various fluorescent proteins such as GFP. Thefollowing are particularly advantageous non-limiting examples of fusionproteins comprising at least one exosomal polypeptide and at least oneFc binding polypeptide, often connected via linkers and optionallyincluding an additional polypeptide or polypeptide domain:

-   -   CD81-Protein A/G CD81 Second loop (as a non-limiting example the        SEQ ID NO 75)    -   CD9-ZZ domain CD9 Second loop (as a non-limiting example the SEQ        ID NO 76)    -   FCAR extracellular domain-2XGGGgSlinker-Lamp2b (as a        non-limiting example the SEQ ID NO 55)    -   FCAR extracellular domain-4XGSlinker-Lamp2b (as a non-limiting        example the SEQ ID NO 54)    -   FCGR1A Extracellular domain-2XGGGgSlinker-Lamp2b (as a        non-limiting example the SEQ ID NO 49)    -   FCGR1A Extracellular domain-4XGSlinker-Lamp2b (as a non-limiting        example the SEQ ID NO 48)    -   FcRN Extracellular domain-4XGSlinker-Lamp2b (as a non-limiting        example the SEQ ID NO 39)    -   FcRN-2XGGGgSlinker-Lamp2b (as a non-limiting example the SEQ ID        NO 40).    -   Gp130 Extracellular domain-2XGGGGS linker-FCAR extracellular        domain-Gp130 transmembrane domain-Leucine Zipper-N terminal        syntenin (as a non-limiting example the SEQ ID NO 52)    -   Gp130 Extracellular domain-2XGGGGS linker-FCGR1A Extracellular        domain-Gp130 transmembrane domain-Leucine Zipper-N terminal        syntenin (as a non-limiting example the SEQ ID NO 46)    -   Gp130 Extracellular domain-2XGGGGS linker-FcRN Extracellular        domain-Gp130 transmembrane domain-Leucine Zipper-N terminal        syntenin (as a non-limiting example the SEQ ID NO 43)    -   Gp130 Extracellular domain-2XGGGGS linker-Z domain-Gp130        transmembrane domain-Leucine Zipper-N terminal syntenin (as a        non-limiting example the SEQ ID NO 34)    -   Transferrin receptor-2XGGGGSlinker-FCAR extracellular domain (as        a non-limiting example the SEQ ID NO 56)    -   Transferrin receptor-2XGGGGSlinker-FCGR1A Extracellular domain        (as a non-limiting example the SEQ ID NO 50)    -   Transferrin receptor-2XGGGGSlinker-FcRN Extracellular domain (as        a non-limiting example the SEQ ID NO 41)    -   Transferrin receptor-2XGGGGSlinker-Z domain (as a non-limiting        example the SEQ ID NO 38)    -   CD63-FCAR extracellular domain CD63 First loop and CD63 Second        loop (as a non-limiting example the SEQ ID NO 53)    -   CD63-FCGR1A Extracellular domain CD63 First loop and CD63 Second        loop (as a non-limiting example the SEQ ID NO 47)    -   CD63-FcRN Extracellular domain CD63 First loop and CD63 Second        loop (as a non-limiting example the SEQ ID NO 44)    -   CD63-Z domain CD63 First loop and CD63 Second loop (as a        non-limiting example the SEQ ID NO 35)    -   TNFR Extracellular domain-2XGGGGS linker-FCAR extracellular        domain-TNFR transmembrane domain-foldon-N terminal syntenin (as        a non-limiting example the SEQ ID NO 51)    -   TNFR Extracellular domain-2XGGGGS linker-FCGR1A Extracellular        domain-TNFR transmembrane domain-foldon-N terminal syntenin (as        a non-limiting example the SEQ ID NO 45)    -   TNFR Extracellular domain-2XGGGGS linker-FcRN Extracellular        domain-TNFR transmembrane domain-foldon-N terminal syntenin (as        a non-limiting example the SEQ ID NO 42)    -   TNFR Extracellular domain-2XGGGGS linker-Z domain-TNFR        transmembrane domain-foldon-N terminal syntenin (as a        non-limiting example the SEQ ID NO 33)    -   Z domain-2XGGGgSlinker-Lamp2b (as a non-limiting example the SEQ        ID NO 37)    -   Z domain-4XGSlinker-Lamp2b (as a non-limiting example the SEQ ID        NO 36)    -   Transferrin receptor—Protein AG (as a non-limiting example the        SEQ ID NO 10 operably fused to as a non-limiting example the SEQ        ID NO 72)

The above-mentioned fusion proteins are merely examples of the manyengineering possibilities the present invention allows for and as suchthey are merely non-limiting embodiments of the present invention. Allcomponents of the fusion proteins as per the present invention may befreely combined, e.g. the fusion proteins may contain one or severalexosomal polypeptides which may be placed C terminally, N terminally, orboth, or anywhere in the fusion protein. Further, the fusion proteinsmay also contain one or several Fc binding polypeptides, which may beplaced C terminally, N terminally, or both, or on one or more of anyloops of e.g. transmembrane parts, or anywhere in the fusion protein.For clarity, more than one type of exosomal polypeptide and more thanone type of Fc binding polypeptide may be comprised in a singleconstruct. Furthermore, additional stretches of amino acids such aslinkers (often comprising the amino acids glycine and serine) and Histags may be included to simplify purification, assaying andvisualization. Also, other peptides and polypeptide domains may also beincluded anywhere in the fusion protein sequence. For instance, variousdomains and regions from various cytokine receptors may advantageouslybe included, for instance various domains of TNFR1, TNFR2, IL17R, IL23R,gp130, IL6R, etc.

In a further embodiment, the Fc binding polypeptides may asabove-mentioned bind to any protein comprising an Fc domain, not onlyantibodies but also other proteins comprising Fc domains, both naturallyoccurring and engineered Fc domain-containing proteins, such as the onesmentioned in several instances above. Advantageously, the presentinvention results in EVs coated with a plurality of proteins comprisingan Fc domain, through interaction between the Fc binding polypeptide andat least one Fc containing protein. The interaction between the Fcbinder and the Fc containing protein is normally based on non-covalentbonds between the Fc binding polypeptide and the Fc domain of the Fccontaining protein. Naturally, one single EV may be coated with morethan one type of Fc domain-containing protein. In one non-limitingexample, the Fc binding EVs are coated with one antibody targeting asuitable target along the PD1 axis, whereas another antibody istargeting a suitable target along the CTLA4 axis. In anothernon-limiting example, the Fc binding EVs are coated with an antibodytargeting an intracellular oncoprotein and a tumor suppressor fused toan Fc domain. One single EV may also, as is typically the case, comprisea substantial plurality of one single type of Fc domain-containingprotein, such as one type of monoclonal antibody. Various combinationsof detection antibodies, targeting antibodies, therapeutic antibodies,Fc containing non-antibody proteins of interest, and antibody-drugconjugates (ADCs (which may be used for diagnostic, imaging andtherapeutic purposes) constitute preferred embodiments of the presentinvention. In advantageous embodiments, the EVs according to the presentinvention are coated with a plurality of proteins comprising an Fcdomain. For instance, when using a highly expressed EV protein such asCD63 or CD81 or syntenin one can achieve very dense coating of thesurface of EVs via the interaction between the Fc binding polypeptideand the Fc domains of the Fc containing proteins. Thus, the presentinvention may be coated with at least 10 proteins comprising an Fcdomain, preferably at least 20 proteins comprising an Fc domain, evenmore preferably at least 30 proteins comprising an Fc domain. Suchproteins may be copies of the same protein (e.g. 50 antibodies targetingan intracellular protein attached on one single EV, by way of example)or more than one protein (e.g. 30 antibodies targeting an intracellularprotein and 40 Fc domain containing tumor suppressor proteins, forinstance). By selecting an optimal combination of EV protein and Fcbinder it may be possibly to increase the display further, in certaincases it may be possible to coat an EV with more than 50 proteinscomprising an Fc domain, or even more than approximately 75 proteinscomprising an Fc domain, or in certain instances even more than 100 Fccontaining proteins per EV.

In a further aspect, the present invention relates to methods forproducing EVs capable of binding to proteins comprising an Fc domain,such as antibodies and proteins engineered to comprise Fc domains. Suchmethods typically comprise the steps of (i) introducing into an EVsource cell a polynucleotide construct which encodes at least one Fcbinding polypeptide and optionally at least one exosomal polypeptide,and (ii) collecting EVs that have been secreted by the EV-producingsource cells, wherein the EVs comprise the Fc binding polypeptide(optionally comprised in a fusion protein) which has been expressed fromthe polynucleotide construct. In a subsequent step, the EVs comprisingthe Fc binding polypeptide may be purified using a suitable purificationtechnique, followed by being exposed to an Fc domain-containing protein,such as an antibody, to enable binding of the Fc containing proteinthrough interaction between the Fc binding polypeptide and the Fc domainof the protein of interest. As above-mentioned, in an alternativeembodiment, step (i) may also include expressing the Fc containingprotein of interest from a polynucleotide construct in the same EVsource cell, thereby achieving endogenous loading of the EV. The Fcbinding polypeptide (optionally comprised in a fusion protein with atleast one exosomal polypeptide) and the Fc containing protein may beexpressed from the same or from different polynucleotide construct,depending on the construct design. In yet another aspect, the presentinvention relates to method for coating EVs with at least one proteincomprising an Fc domain, such as an antibody. Such methods comprise thesteps of (i) providing an EV comprising at least one Fc bindingpolypeptide comprising at least one Fc binding protein, optionally fusedto at least one exosomal polypeptide, and (ii) allowing the Fc bindingpolypeptide to bind the Fc domain of at least one protein comprising anFc domain. The EV source cells used for production of EVs comprising theFc binding polypeptides may be either stably or transiently transfectedwith the polynucleotide construct needed to generate the EVs carryingthe Fc binding polypeptide-Fc containing protein complex. Stabletransfection is advantageous as it enables creation of master cell banks(MCBs) and working cell banks (WCBs). However, transient transfection isalso advantageous in certain instances, for example when assessingdifferent constructs or e.g. when rapidly creating an autologous therapycomprising EVs obtained from a patient's own EV-producing cells. Thesemethods may be followed by additional steps to optimize the EVs and theFc containing proteins for application in research, diagnostics, imagingand therapy. For instance, the EVs comprising Fc binding polypeptidesmay be formulated in a liquid formulation in a suitable vessel forstorage and use. Such a liquid formulation may be frozen, lyophilized,formulated in a lipid or protein formulation, and/or handled in anysuitable way depending on the application in question. The Fc containingprotein may also be formulated as a liquid formulation but alternativelycan also be formulated in another physical form, for instance as alyophilized powder. Importantly, the different components of thedetection kits and the kits of parts shall be designed and produced withthe end application in mind, be it flow cytometry, fluorescencemicroscopy, electron microscopy, in vitro, in vivo, and/or ex vivodelivery methods, etc.

In a further aspect, the present invention pertains to the use of EVs asdelivery vehicles for antibodies and other Fc domain containing proteinsof interest, in vitro, ex vivo and/or in vivo. Furthermore, the presentinvention also provides for the use of EVs attached to Fc containingproteins as delivery vehicles for other diagnostic, imaging, and/ortherapeutic agents of interest. As a non-limiting example, EVs havingattached to their surface a targeting antibody may also containadditional diagnostic and/or therapeutic agents which may be presenteither inside the EV and/or in the EV membrane. For instance, anantibody-coated EV may be used to deliver an RNA therapeutics cargo(such as an mRNA, an siRNA, an oligonucleotide, etc.) to a target cell,tissue and/or organ. In another non-limiting example, EVs havingattached to their surface an Fc containing targeting protein (such as ascFv which have been engineered to contain an Fc domain) and comprisinginside the EV or in/on the EV membrane a therapeutic protein such asCas9 for gene editing, optionally together with a CRISPR RNA guidestrand.

In further aspects, the methods of the present invention may alsocomprise exposing the EV source cells to serum starvation, hypoxia,bafilomycin, or cytokines such as TNF-alpha and/or IFN-gamma, in orderto influence the yield or properties of the resulting EVs. The EVproduction scale and timeline will be heavily dependent on theEV-producing cell or cell line and may thus be adapted accordingly by aperson skilled in the art. The methods as per the present invention mayfurther comprise an EV purification step, which may be carried out priorto co-incubating the EVs comprising the Fc binding polypeptide with theFc domain-containing protein (such as an antibody) to be attached to theEVs. EVs may be purified through a procedure selected from a group oftechniques comprising liquid chromatography (LC), high-performanceliquid chromatography (HPLC), bead-eluate chromatography, spinfiltration, tangential flow filtration (TFF), hollow fiber filtration,centrifugation, immunoprecipitation, flow field fractionation, dialysis,microfluidic-based separation, etc., or any combination thereof. In anadvantageous embodiment, the purification of the EVs is carried outusing a sequential combination of filtration (preferably ultrafiltration(UF), tangential flow filtration or hollow fiber filtration) and sizeexclusion liquid chromatography (LC) or bead-eluate chromatography. Thiscombination of purification steps results in optimized purification,which in turn leads to superior activity and potency in variousapplications, assays and analytical methods. Further, as compared toultracentrifugation (UC), which is routinely employed for purifyingexosomes, sequential filtration-chromatography is considerably fasterand possible to scale to higher manufacturing volumes, which is asignificant drawback of the current UC methodology that dominates theprior art. Another advantageous purification methodology is tangentialflow filtration (TFF), which offers scalability and purity, and whichmay be combined with any other type of purification technique.

In yet another aspect, the present invention pertains to pharmaceutical,diagnostic, and/or imaging compositions comprising EVs, normally in theform of populations of EVs, as per the present invention. Typically, thecompositions as per the present invention comprise one type of EVs (i.e.a population of EVs comprising a certain type of Fc binding protein andhaving attached to it one or more types of Fc containing proteins, suchas antibodies) formulated with at least one acceptable excipient.However, more than one type of EV population may naturally be comprisedin a single composition, for instance in cases where a combination ofantibodies is desirable. Naturally however, as above-mentioned, a singleEV or a single population of EVs may comprise more than oneFc-containing protein (e.g. an antibody) bound to the EV surface. Theselection of suitable excipients is determined by the actualapplication, and excipients may be selected from the group comprisingany pharmaceutically acceptable and/or research grade material,composition or vehicle, for instance a solid or liquid filler, adiluent, an excipient, a carrier, a solvent or an encapsulatingmaterial, which may be involved in e.g. suspending, maintaining theactivity of or carrying or transporting the EV population.

In yet another aspect, the present invention relates to EVs as per thepresent invention for use in medicine, for instance in imaging,diagnostics, and/or therapy or theranostics. Naturally, when the EVs asper the present invention are used in medicine, it is in fact normally apopulation of EVs that is being used. The dose of EVs administered to apatient will depend on the number of e.g. Fc containing proteins ofinterest that has been coated on the EV surface, the disease or thesymptoms to be detected, diagnosed, treated or alleviated, theadministration route, the mechanism of action of the any additionaldiagnostic, imaging and/or therapeutic agent itself, the inherentproperties of the EVs, the presence of any targeting antibodies or othertargeting entities, as well as various other parameters of relevanceknown to a skilled person.

The EVs of the present invention carrying the Fc containing proteins maybe used for several different diagnostic, imaging, and/or therapeuticaspects. In one embodiment, the EVs are covered with antibodies or otherFc containing proteins that target a specific cell type, tissue, and/ororgan. This is a highly powerful way of targeting EVs, which maycomprise other imaging agents or pharmaceutical agents in addition tothe Fc containing protein, to tissues of interest, and could represent astep chance in theranostics, diagnostics, imaging, and drug delivery. Inanother embodiment, antibodies or other Fc domain-containing proteinsthat interact with a target antigen of interest can be efficientlydelivered to tissues of interest that are typically hard to reach, usingEVs with ability to cross biological barriers. This approach may forinstance enable delivery of monoclonal antibodies into the centralnervous system or into the brain for both therapy and diagnosis/imaging.In yet another embodiment, coating of EVs with Fc containing proteins isa way of multiplexing the Fc containing protein of interest, in order toenhance or influence its target avidity or the conformation of itsbinding to a target of interest in vitro, ex vivo and in vivo. In yetanother embodiment, the EVs as per the present invention enable improveddelivery and efficacy of antibody-drug conjugates (ADCs) orreceptor-drug conjugates, as multiplexing of ADCs may significantlyenhance their therapeutic activity and their presence on EVs means theycan also enter target cells. In a further embodiment, the ability of EVsto enter target cells means that the EVs of the present invention opensup the entire intracellular space and make it druggable by essentiallyany protein comprising an Fc domain and/or any protein onto which an Fcdomain can be fused (such as an enzyme for enzyme replacement therapy,nuclease such as Cas9, or a tumor suppressor such as any one of p53,pVHL, APC, CD95, ST5, YPEL3, ST7, and ST14).

The EVs and the EV populations thereof as per the present invention maythus be used for diagnostic, imaging, research, prophylactic and/ortherapeutic purposes, e.g. for use in the diagnosis, imaging,evaluation, prophylaxis and/or treatment and/or alleviation of variousdiseases and disorders. A non-limiting sample of diseases wherein theEVs as per the present invention may be applied comprises Crohn'sdisease, ulcerative colitis, ankylosing spondylitis, rheumatoidarthritis, multiple sclerosis, systemic lupus erythematosus,sarcoidosis, idiopathic pulmonary fibrosis, psoriasis, tumor necrosisfactor (TNF) receptor-associated periodic syndrome (TRAPS), deficiencyof the interleukin-1 receptor antagonist (DIRA), endometriosis,autoimmune hepatitis, scleroderma, myositis, stroke, acute spinal cordinjury, vasculitis, Guillain-Barré syndrome, acute myocardialinfarction, ARDS, sepsis, meningitis, encephalitis, liver failure,non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease(NAFLD), kidney failure, heart failure or any acute or chronic organfailure and the associated underlying etiology, graft-vs-host disease,Duchenne muscular dystrophy and other muscular dystrophies, lysosomalstorage diseases such as Gaucher disease, Fabry's disease, MPS I, II(Hunter syndrome), and III, Niemann-Pick disease, Pompe disease, etc.,neurodegenerative diseases including Alzheimer's disease, Parkinson'sdisease, Huntington's disease and other trinucleotide repeat-relateddiseases, dementia, ALS, cancer-induced cachexia, anorexia, diabetesmellitus type 2, and various cancers. Virtually all types of cancer arerelevant disease targets for the present invention, for instance, Acutelymphoblastic leukemia (ALL), Acute myeloid leukemia, Adrenocorticalcarcinoma, AIDS-related cancers, AIDS-related lymphoma, Anal cancer,Appendix cancer, Astrocytoma, cerebellar or cerebral, Basal-cellcarcinoma, Bile duct cancer, Bladder cancer, Bone tumor, Brainstemglioma, Brain cancer, Brain tumor (cerebellar astrocytoma, cerebralastrocytoma/malignant glioma, ependymoma, medulloblastoma,supratentorial primitive neuroectodermal tumors, visual pathway andhypothalamic glioma), Breast cancer, Bronchial adenomas/carcinoids,Burkitt's lymphoma, Carcinoid tumor (childhood, gastrointestinal),Carcinoma of unknown primary, Central nervous system lymphoma,Cerebellar astrocytoma/Malignant glioma, Cervical cancer, Chroniclymphocytic leukemia, Chronic myelogenous leukemia, Chronicmyeloproliferative disorders, Colon Cancer, Cutaneous T-cell lymphoma,Desmoplastic small round cell tumor, Endometrial cancer, Ependymoma,Esophageal cancer, Extracranial germ cell tumor, Extragonadal Germ celltumor, Extrahepatic bile duct cancer, Eye Cancer (Intraocular melanoma,Retinoblastoma), Gallbladder cancer, Gastric (Stomach) cancer,Gastrointestinal Carcinoid Tumor, Gastrointestinal stromal tumor (GIST),Germ cell tumor (extracranial, extragonadal, or ovarian), Gestationaltrophoblastic tumor, Glioma (glioma of the brain stem, CerebralAstrocytoma, Visual Pathway and Hypothalamic glioma), Gastric carcinoid,Hairy cell leukemia, Head and neck cancer, Heart cancer, Hepatocellular(liver) cancer, Hodgkin lymphoma, Hypopharyngeal cancer, IntraocularMelanoma, Islet Cell Carcinoma (Endocrine Pancreas), Kaposi sarcoma,Kidney cancer (renal cell cancer), Laryngeal Cancer, Leukemias ((acutelymphoblastic (also called acute lymphocytic leukemia), acute myeloid(also called acute myelogenous leukemia), chronic lymphocytic (alsocalled chronic lymphocytic leukemia), chronic myelogenous (also calledchronic myeloid leukemia), hairy cell leukemia)), Lip and Oral, CavityCancer, Liposarcoma, Liver Cancer (Primary), Lung Cancer (Non-SmallCell, Small Cell), Lymphomas, AIDS-related lymphoma, Burkitt lymphoma,cutaneous T-Cell lymphoma, Hodgkin lymphoma, Non-Hodgkin,Medulloblastoma, Merkel Cell Carcinoma, Mesothelioma, MetastaticSquamous Neck Cancer with Occult Primary, Mouth Cancer, MultipleEndocrine Neoplasia Syndrome, Multiple Myeloma/Plasma Cell Neoplasm,Mycosis Fungoides, Myelodysplastic/Myeloproliferative Diseases,Myelogenous Leukemia, Chronic Myeloid Leukemia (Acute, Chronic),Myeloma, Nasal cavity and paranasal sinus cancer, Nasopharyngealcarcinoma, Neuroblastoma, Oral Cancer, Oropharyngeal cancer,Osteosarcoma/malignant fibrous histiocytoma of bone, Ovarian cancer,Ovarian epithelial cancer (Surface epithelial-stromal tumor), Ovariangerm cell tumor, Ovarian low malignant potential tumor, Pancreaticcancer, Pancreatic islet cell cancer, Parathyroid cancer, Penile cancer,Pharyngeal cancer, Pheochromocytoma, Pineal astrocytoma, Pineal germinoma, Pineoblastoma and supratentorial primitive neuroectodermaltumors, Pituitary adenoma, Pleuropulmonary blastoma, Prostate cancer,Rectal cancer, Renal cell carcinoma (kidney cancer), Retinoblastoma,Rhabdomyosarcoma, Salivary gland cancer, Sarcoma (Ewing family of tumorssarcoma, Kaposi sarcoma, soft tissue sarcoma, uterine sarcoma), Sezarysyndrome, Skin cancer (nonmelanoma, melanoma), Small intestine cancer,Squamous cell, Squamous neck cancer, Stomach cancer, Supratentorialprimitive neuroectodermal tumor, Testicular cancer, Throat cancer,Thymoma and Thymic carcinoma, Thyroid cancer, Transitional cell cancerof the renal pelvis and ureter, Urethral cancer, Uterine cancer, Uterinesarcoma, Vaginal cancer, Vulvar cancer, Waldenström macroglobulinemia,and/or Wilm's tumor.

The EVs as per the present invention may be administered to a human oranimal subject via various different administration routes, for instanceauricular (otic), buccal, conjunctival, cutaneous, dental,electro-osmosis, endocervical, endosinusial, endotracheal, enteral,epidural, extra-amniotic, extracorporeal, hemodialysis, infiltration,interstitial, intra-abdominal, intra-amniotic, intra-arterial,intra-articular, intrabiliary, intrabronchial, intrabursal,intracardiac, intracartilaginous, intracaudal, intracavernous,intracavitary, intracerebral, intracisternal, intracorneal, intracoronal(dental), intracoronary, intracorporus cavernosum, intradermal,intradiscal, intraductal, intraduodenal, intradural, intraepidermal,intraesophageal, intragastric, intragingival, intraileal, intralesional,intraluminal, intralymphatic, intramedullary, intrameningeal,intramuscular, intraocular, intraovarian, intrapericardial,intraperitoneal, intrapleural, intraprostatic, intrapulmonary,intrasinal, intraspinal, intrasynovial, intratendinous, intratesticular,intrathecal, intrathoracic, intratubular, intratum or, intratym panic,intrauterine, intravascular, intravenous, intravenous bolus, intravenousdrip, intraventricular, intravesical, intravitreal, iontophoresis,irrigation, laryngeal, nasal, nasogastric, occlusive dressing technique,ophthalmic, oral, oropharyngeal, other, parenteral, percutaneous,periarticular, peridural, perineural, periodontal, rectal, respiratory(inhalation), retrobulbar, soft tissue, subarachnoid, subconjunctival,subcutaneous, sublingual, submucosal, topical, transdermal,transmucosal, transplacental, transtracheal, transtympanic, ureteral,urethral, and/or vaginal administration, and/or any combination of theabove administration routes, which typically depends on the disease tobe treated and/or the characteristics of the antibody or the EVpopulation as such.

Suitable targeting antibodies in accordance with the present inventionmay target antigens deriving from e.g. tumors, solid organs, bodilystructures, cell or tissue types. Non-limiting examples of the origin ofantigens that may be targeted by targeting antibodies include liver,lung, kidney, heart, pancreas, adrenal glands, thyroid glands,parathyroid glands, brain including all brain regions (for instancethalamus, hypothalamus, striatum, etc.), the blood-brain-barrier, theCNS, the PNS, bone marrow, the skin, the vascular system, the lymphaticsystem including the spleen, joints, eyes, muscle tissues, sites ofinflammation, sites of injury, and cell types such as adipocytes, musclecells (myoblasts and myotubes), satellite cells, cardiac cells,endothelial cells, fibroblasts, hepatocytes, renal cells, pericytes,neurons, glia cells, astrocytes, oligodendrocytes, macrophages,DC-cells, B-cells, T-cells, NK-cells, chrondrocytes, osteoblast,osteocytes, epithelial cells, erythrocytes, earlier progenitors such asmultipotential hematopoietic stem cells/hemocytoblasts, myeloidprogenitors, lymphoid progenitors, etc. Non-limiting examples ofantigens relevant for targeting cancer includes adenocarcinoma antigen,alpha-fetoprotein, BAFF, C242 antigen, CA-125, carbonic anhydrase 9(CA-IX), disialoganglioside (GD2), 4-IBB, 5T4, CD22, CD221, CD23 (IgEreceptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52,CD56, CD74, CD80, CEA, CNT0888, CTLA-4, DR5, EGFR, EpCAM, CD3, FAP,fibronectin extra domain-B, folate receptor 1, GD2, GD3 ganglioside,glycoprotein 75, GPNMB, HER2/neu, HGF, human scatter factor receptorkinase, IGF-1 receptor, IGF-I, IgGI, LI-CAM, IL-13, IL-6, insulin-likegrowth factor I receptor, integrin α5β1, integrin αvβ3, legumain,MORAb-009, MS4A1, MUC1, mucin CanAg, C-MET, CCR4, CD 152, CD 10, CD 19,CD20, CD200, N-glycolylneuraminic acid, NPC-IC, PDGF-R a, PDL192,phosphatidylserine, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2,vimentin, RANKL, RON, ROR1, SCH 900105, SDC1, SLAMF7, TAG-72, tenascinC, TGF beta 2, TGF-β, TRAIL-R1, TRAIL-R2, folic acid receptor,transferrin receptors and any combination thereof.

In a further embodiment, suitable non-limiting examples of antibodies inaccordance with the present invention may be any one or more ofAbagovomab, Abciximab, Actoxumab, Adalimumab, Adecatumumab,Adotrastuzumab emtansine, Aducanumab, Afelimomab, Afutuzumab, AlacizumabAlemtuzumab, Alirocumab, Altumomab pentetate, Amatuximab, Anatumomabmafenatox, Anifrolumab, Anrukinzumab, Apolizumab, Arcitumomab,Aselizumab, Atezolizumab, Atinumab, Atlizumab, Atorolimuma, Avelumab,Bapineuzumab, Basiliximab, Bavituximab, Bectumomab, Belimumab,Benralizumab, Bertilimumab, Besilesomab, Bevacizumab, Bezlotoxumab,Biciromab, Bimagrumab, Bivatuzumab mertansine, Blinatumomab, Blosozumab,Brentuximab vedotin, Briakinumab, Brodalumab, Canakinumab, Cantuzumabmertansine, Cantuzumab ravtansine, Caplacizumab, Capromab pendetide,Carlumab, Catumaxomab, cBR96-doxorubicin immunoconjugate, Cedelizumab,Certolizumab pegol, Cetuximab, Citatuzumab bogatox, Cixutumumab,Clazakizumab, Clenoliximab, Clivatuzumab tetraxetan, Conatumumab,Concizumab, Crenezumab, CR6261, Dacetuzumab, Daclizumab, Dalotuzumab,Daratumumab, Demcizumab, Denosumab, Detumomab, Dinutuximab, Dorlimomabaritox, Drozitumab, Duligotumab, Dupilumab, Durvalumab, Dusigitumab,Ecromeximab, Eculizumab, Edobacomab, Edrecolomab, Efalizumab, Efungumab,Eldelumab, Elotuzumab, Elsilimomab, Enavatuzumab, Enlimomab pegol,Enokizumab, Enoticumab, Ensituximab, Epitumomab cituxetan, Epratuzumab,Erlizumab, Ertumaxomab, Etanercept, Etaracizumab, Etrolizumab,Evolocumab, Exbivirumab, Fanolesomab, Faralimomab, Farletuzumab,Fasinumab, FBTA05, Felvizumab, Fezakinumab, Ficlatuzumab, Figitumumab,Flanvotumab, Fontolizumab, Foralumab, Foravirumab, Fresolimumab,Fulranumab, Futuximab, Galiximab, Ganitumab, Gantenerumab, Gavilimomab,Gemtuzumab ozogamicin, Gevokizumab, Girentuximab, Glembatumumab vedotin,Golimumab, Gomiliximab, Guselkumab, Ibalizumab, Ibritumomab tiuxetan,Icrucumab, Igovomab, IMAB362, Imciromab, Imgatuzumab, Inclacumab,Indatuximab ravtansine, Infliximab, Intetumumab, Inolimomab, Inotuzumabozogamicin, Ipilimumab, Iratumumab, Itolizumab, Ixekizumab, Keliximab,Labetuzumab, Lambrolizumab, Lampalizumab, Lebrikizumab, Lemalesomab,Lerdelimumab, Lexatumumab, Libivirumab, Ligelizumab, Lintuzumab,Lirilumab, Lodelcizumab, Lorvotuzumab mertansine, Lucatumumab,Lumiliximab, Mapatumumab, Margetuximab, Maslimomab, Mavrilimumab,Matuzumab, Mepolizumab, Metelimumab, Milatuzumab, Minretumomab,Mitumomab, Mogamulizumab, Morolimumab Motavizumab, Moxetumomabpasudotox, Muromonab-CD3, Nacolomab tafenatox, Nam ilumab, Naptumomabestafenatox, Narnatumab, Natalizumab, Nebacumab, Necitumumab,Nerelimomab, Nesvacumab, Nimotuzumab, Nivolumab, Nofetumomab merpentan,Ocaratuzumab, Ocrelizumab, Odulimomab, Ofatumumab, Olaratumab,Olokizumab, Omalizumab, Onartuzumab, Ontuxizumab, Oportuzumab monatox,Oregovomab, Orticumab, Otelixizumab, Otlertuzumab, Oxelumab, Ozanezumab,Ozoralizumab, Pagibaximab, Palivizumab, Panitumumab, Pankomab,Panobacumab, Parsatuzumab, Pascolizumab, Pateclizumab, Patritumab,Pembrolizumab, Pemtumomab, Perakizumab, Pertuzumab, Pexelizumab,Pidilizumab, Pinatuzumab vedotin, Pintumomab, Placulumab, Polatuzumabvedotin, Ponezumab, Priliximab, Pritoxaximab, Pritumumab, PRO 140,Quilizumab, Racotumomab, Radretumab, Rafivirumab, Ramucirumab,Ranibizumab, Raxibacumab, Regavirumab, Reslizumab, Rilotumumab,Risankizumab, Rituximab, Robatumumab, Roledumab, Romosozumab,Rontalizumab, Rovelizumab, Ruplizumab, Samalizumab, Sarilumab, Satumomabpendetide, Secukinumab, Seribantumab, Setoxaximab, Sevirumab,Sibrotuzumab, Sifalimumab, Siltuximab, Simtuzumab, Siplizumab,Sirukumab, Solanezumab, Solitomab, Sonepcizumab, Sontuzumab, Stamulumab,Sulesomab, Suvizumab, Tabalumab, Tacatuzumab tetraxetan, Tadocizumab,Talizumab, Tanezumab, Taplitumomab paptox, Tefibazumab, Telimomabaritox, Tenatumomab, Teneliximab, Teplizumab, Teprotumumab, Ticilimumab,Tildrakizumab, Tigatuzumab, Tocilizumab, Toralizumab, Tositumomab,Bexxar, Tovetumab, Tralokinumab, Trastuzumab, Tregalizumab,Tremelimumab, Tucotuzumab celmoleukin, Tuvirumab, Ublituximab, Urelumab,Urtoxazumab, Ustekinumab, Vantictumab, Vapaliximab, Vatelizumab,Vedolizumab, Veltuzumab, Vepalimomab, Vesencumab, Visilizumab,Volociximab, Vorsetuzumab mafodotin, Votumumab, Zalutumumab,Zanolimumab, Zatuximab, Ziralimumab, Zolimomab aritox, or anycombination thereof. Importantly, the present invention provides for thedelivery of any antibody, regardless of whether its target isintracellular and/or extracellular. The ability of the EVs of thepresent invention to efficiently internalize antibodies represent astep-change in monoclonal antibody development and may enable targetingantigens and targets across the entire intracellular compartment oftarget cells. Importantly, antibodies and Fc containing proteins as perthe present invention may advantageously be labelled for detection andimaging purposes, for instance using fluorophores, MRI agents, PETagents, radioactive agents, enzymes, nanoparticles, metals, organic andinorganic compounds, etc.

It shall be understood that the above described exemplifying aspects,embodiments, alternatives, and variants can be modified withoutdeparting from the scope of the invention. The invention will now befurther exemplified with the enclosed examples, which naturally also canbe modified considerably without departing from the scope and the gistof the invention.

Experimental Part Materials and Methods

Construct design and cloning: Various protein constructs comprising atleast one Fc binding polypeptide and optionally at least one exosomalpolypeptide have been constructed, cloned into vectors and produced inseveral different EV-producing cell sources. ORFs were typicallygenerated by synthesis and cloned into the mammalian expression vectorpSF-CAG-Amp. Briefly, synthesized DNA and vector plasmid were digestedwith enzymes Notl and Sall as per manufacturers instruction (NEB).Restricted, purified DNA fragments were ligated together using T4 ligaseas per manufacturers instruction (NEB). Successful ligation events wereselected for by bacterial transformation on ampicillin-supplementedplates. Plasmid for transfection was generated by ‘maxi-prep’, as permanufacturers instruction.

In cases where Fc containing proteins were endogenously produced in thesame EV-producing cell source that expresses the fusion protein, ORFsequences were purchased (Origene Technologies, Inc.) and amplified andcloned into the MSC A site of pIRES bicistronic expression vector(Clonetech, Laboratories Inc.) such that upon translation the optionalexosomal polypeptide was fused to the Fc binding polypeptide in oneconstruct, whereas the Fc containing protein of interest was translatedseparately (from a separate construct or from the same construct) andtransported into the EV to be formed in the EV-producing cell source.Most of the cloning was performed using the NEBuilder HiFi DNA AssemblyCloning Kit (NEB, Inc.) and confirmed using Sanger sequencing (SourceBioScience). The pIRES vector enables bicistronic expression of bothtransgenes simultaneously, ensuring EV-producing cells would expressboth the Fc binding polypeptide (optionally as a fusion protein) and theFc containing protein of interest simultaneously. Plasmids weretransformed into the NEB 5-alpha Competent E. coli cells (NEB, Inc.) andgrown overnight in a shaking incubator according to manufacturer'srecommendations. Plasm ids were isolated and purified using the‘maxi-prep plasmid DNA purification kit’ (Qiagen), as per manufacturer'sinstruction.

Cell Culture and Transfection

Depending on the experimental design and assays, in certain cases,non-viral transient transfection and exosome production was carried outin conventional 2D cell culture, whereas in other cases virus-mediatedtransduction was employed to create stable cell lines, which weretypically cultured in bioreactors of different type. For conciseness,only a few examples are mentioned herein.

HEK293T cells were typically seeded into 15 cm dishes (9×10⁶ cells perdish) and left overnight in serum-containing DMEM as recommended byATCC. The following day the cells were transiently transfected withlipoplexed DNA added directly onto cells. Briefly, DNA andpolyethyleneimine (PEI) were separately incubated in OptiMEM for 5minutes before combining together for 20 minutes at room temperature.Lipoplexed DNA and cells were co-incubated for 6 hours following whichconditioned culture media was changed to OptiMEM for 48 hours. Othercells and cell lines that were evaluated in dishes, flasks and othercell culture vessels included bone marrow-derived mesenchymal stromalcells (BM-MSCs) and Wharton's jelly-derived MSCs (WJ-MSCs), amnioncells, fibroblasts, various endothelial and epithelial cells, as well asvarious immune cells and cell lines.

In the case of viral transduction and creation of stable cell lines forvarious combinations of Fc binding polypeptides (optionally comprised infusion proteins with exosomal polypeptides) and Fc containing proteinsof interest, cell sources such as BM-MSCs, WJ-MSC, fibroblasts, amnioncells, fibroblasts, various endothelial and epithelial cells, werevirus-transduced, typically using lentivirus (LV). Typically, 24 hoursbefore infection, 100.000 cells (e.g. fibroblasts, MSCs, etc.) or200.000 cells (e.g. HEK293T) are plated in a 6-well plate. 2 uL of LVand optionally Polybrene (or hexadimethrine bromide, final concentrationon the well of 8 ug/mL) are added, and 24 hours post transduction thecell medium of transduced cells is changed to fresh complete media. At72 hours post transduction, puromycin selection (4-6 μg/ml) isperformed, normally for 7 days followed by analysis of stable expressionof the Fc binding polypeptide (optionally as a fusion protein togetherwith an EV protein).

Stable cells were cultured in either 2D culture or in bioreactors,typically hollow-fiber bioreactors or stir-rank bioreactors, andconditioned media was subsequently harvested for exosome preparation.Various preparation and purification steps were carried out. Thestandard workflow comprises the steps of pre-clearing of thesupernatant, filtration-based concentration, chromatography-basedremoval of protein contaminants, and optional formulation of theresultant exosome composition in a suitable buffer for in vitro, ex vivoand/or in vivo assays.

Assays and Analytics

Western blot is a highly convenient analytical method to evaluate theenrichment of fusion proteins in EVs. Briefly, SDS-PAGE was performedaccording to manufacturer's instruction (Invitrogen, Novex PAGE 4-12%gels), whereby 1×10¹⁰ exosomes and 20 ug cell lysate were loaded perwell. Proteins from the SDS-PAGE gel were transferred to PVDF membraneaccording to manufacturer's instruction (Immobilon, Invitrogen).Membranes were blocked in Odyssey blocking buffer (Licor) and probedwith antibodies against the Fc binding polypeptide and/or the exosomalprotein according to supplier's instruction (Primary antibodies—Abcam,Secondary antibodies—Licor). Molecular probes visualized at 680 and 800nm wavelengths.

For EV size determination, nanoparticle tracking analysis (NTA) wasperformed with a NanoSight instrument equipped with analytical software.For all recordings, we used a camera level of 13 or 15 and automaticfunction for all post-acquisition settings. Electron microscopy andfluorescence microscopy were frequently used to understand intracellularlocation and release and to quantitate and analyze EVs and EVs coatedwith labelled Fc containing proteins, e.g. antibodies.

EVs were isolated and purified using a variety of methods, typically acombination of filtration such as TFF and LC, in particular bead-eluteLC. Typically, EV-containing media was collected and subjected to a lowspeed spin at 300 g for 5 minutes, followed by 2000 g spin for 10minutes to remove larger particles and cell debris. The supernatant wasthen filtered with a 0.22 μm syringe filter and subjected to differentpurification steps. Large volumes were diafiltrated and concentrated toroughly 20 ml using the Vivaflow 50R tangential flow (TFF) device(Sartorius) with 100 kDa cutoff filters or the KR2i TFF system (SpectrumLabs) with 100 or 300 kDa cutoff hollow fibre filters. Thepreconcentrated medium was subsequently loaded onto the bead-eluatecolumns (HiScreen or HiTrap Capto Core 700 column, GE Healthcare LifeSciences), connected to an ÄKTAprime plus or ÄKTA Pure 25 chromatographysystem (GE Healthcare Life Sciences). Flow rate settings for columnequilibration, sample loading and column cleaning in place procedurewere chosen according to the manufacturer's instructions. The sample wascollected according to the UV absorbance chromatogram and concentratedusing an Amicon Ultra-15 10 kDa molecular weight cut-off spin-filter(Millipore) to a final volume of 100 μl and stored at −80° C. forfurther downstream analysis. To assess the protein and RNA elutionprofiles, media was concentrated and diafiltrated with KR2i TFF systemusing 100 kDa and 300 kDa hollow fibre filters and a sample analysed ona Tricorn 10/300 Sepharose 4 Fast Flow (S4FF) column (GE Healthcare LifeSciences).

EXAMPLES Example 1: Binding of IgG to EVs Comprising Fc BindingPolypeptides (Fc-Binding EVs)

EVs were isolated from the conditioned medium from engineered HEK293Tcells (control versus Fc-binding construct that stably express Gp130Extracellular domain-2XGGGGS linker-Z domain-Gp130 transmembranedomain-Leucine Zipper-N terminal syntenin-His tag) using tangential flowfiltration with 300 kd hollow fiber columns, followed by ultrafiltrationusing 10 kd spin filters for concentration. The binding capacity for IgGby the Fc-binding EVs were then assessed using electron microscopy andflow cytometry.

For electron microscopy, 1×10{circumflex over ( )}9 EVs were incubatedwith Rabbit anti-goat 10 nm antibody conjugated with gold Nanoparticlesfor 2 h at 37° C. As shown in FIG. 2 , Fc-binding EVs (A) are decoratedwith nanogold labeled antibodies (i.e. Fc containing proteins), whereascontrol EVs (B) do not have any antibodies bound.

For flow cytometry, 1×10{circumflex over ( )}8 EVs were incubatedovernight on an orbital shaker at 450 rpm for 16 hours in 120 μl PBSwith 15 μl antibody-coated capture beads from the MACSPlex Exosome Kit,human (Miltenyi Biotec, Order no 130-108-813). After washing, 3 μg ofAlexaFluor647-conjugated human IgG Fc fragments (Jackson Laboratories,Catalogue 009-600-008) were added to controls without EVs (A), controlEVs (B), or Fc-binding EVs (Synthenin-gp130-zDomain-gp130) (C). After 1hour incubation at room temperature, unbound Fc fragments were washedaway and samples were analyzed via flow cytometry. In FIG. 3 respectiveleft dotplots show hard-dyed capture bead populations using B1-A(Excitation: 488 nm, Emission Filter: 500-550 nm; Area) versus B2-A(Excitation: 488 nm, Emission Filter: 565-605 nm, Area) parameters.Respective right plots show R1-A (Excitation: 635 nm, Emission Filter:655-730 nm, Area) versus B2-A parameters, demonstrating binding ofAlexaFluor647 labeled Fc-Fragments to EVs which have bound to thecapture beads only in (C). FIG. 3 shows that the Fc-binding EVs bindboth to AlexaFluor647-labelled Fc fragments (human IgG) and veryefficiently also to the Fc domains of all 39 different antibodies whichare coated on all capture bead populations by the manufacturer includedin the kit, including the two negative control bead populations.

These results demonstrate that Fc-binding EVs can be used for thepurpose of standardization, positive control, calibration control orcompensation control in any kind of assay, method or application thatcomprises the binding of antibodies or Fc containing proteins or probesto EVs. Normally, the antibodies or Fc containing proteins will belabelled with a fluorophore or other kind of detection moiety. Since thesignals expected from the Fc-mediated binding of antibodies or Fccontaining proteins normally will be at least as high as a signal thatwould be expected from the specific complementary determining region(CDR)-mediated binding of an antibody to an EV, these Fc-binding EVswill represent a suitable positive control for any kind of assay, e.g.flow cytometry based phenotyping of EVs, and are also compatible withany kind of antibody that might be used in flow cytometry or for thatmatter for any other type of technique or assay (such as fluorescencemicroscopy, nanoimaging, nanoparticle tracking analysis, dynamic lightscattering, electron microscopy using antibodies attached to a e.g. agold nanoparticle, etc.). This especially will be helpful formulti-color assays that aim to combine different types of antibodieswith different fluorescent labels, e.g. antibody 1 labelled with FITCand antibody 2 labelled with PE. In such an multi-color approach mostmethods require compensation for spectral overlaps between the emittedfluorescence signals of respective antibodies, and thus positivecontrols are needed in order to perform this spectral compensation,which can be generated by labellin Fc binding EVs with respectiveantibodies. In terms of calibration of instruments or reagents orstandardization of assays, an EV entity that binds a given antibody orFc-containing probe will also be suitable to monitor variation and tovalidate the complete setup over time, e.g. as a stable source ofcalibration in a diagnostics test or laboratory setting.

Example 2: FCGR1A Lamp2B EVs for Delivery of Anti-HER2 Antibody

EVs were isolated from HEK293T cells (either stably expressing FCGR1AExtracellular domain-4XGSlinker-Lamp2b, the Fc binder FCGR1A alone, ortheir wild type controls) using ultrafiltration and size exclusionchromatography. EVs were labelled with PKH26 red fluorescent dye, anddecorated with anti-HER2 antibody or its isotype control byco-incubating EVs and antibody for 1 h at 37° C. Unbound antibody wasremoved by size exclusion chromatography. Uptake of antibody decoratedEVs was characterized in HER2 low-expressing cell line MDA-MB-231 and inHER2 high-expressing cell line MDA-MB-361 using flow cytometry. FIG. 4shows that anti-HER2 antibody increases uptake of decorated EVs ascompared to isotype control decorated and wild type EVs only in HER2high-expressing cell line MDA-MB-361, but not in HER2 low-expressingcell line MDA-MB-231. The EVs expressing FCGR1A alone (not comprised ina fusion protein) displayed similar activity, and similar results werealso obtained with EVs expressing CD63-ZZ fusion proteins.

Example 3: Intracellular Uptake and Delivery of Antibodies ViaFc-Binding EVs

EVs were isolated from the conditioned medium of Wharton's jelly-derivedMSCs (either stably expressing TNFR Extracellular domain-2XGGGGSlinker-Z domain-TNFR transmembrane domain-foldon-N terminal synteninfusion proteins or control) using ultrafiltration and size exclusionchromatography. In order to investigate whether Fc-binding EVs can beemployed for intracellular delivery of Abs, 4×10{circumflex over ( )}11EVs were incubated in 400 μl for 16 hours (overnight) with total 3 μgAlexaFluor488-labelled anti-Lamin B2 IgGs [abcam ab200426, Rabbitmonocolonal EPR9701(B)]. For the uptake experiment, Huh7 cells wereplated in 48 well plates at 30,000 cells per well and incubated for 16hours before 0.675×10{circumflex over ( )}11 antibody-labelled EVs wereadded. Cells were incubated for 2 hours at 37° C. and 5% CO₂ in ahumidified atmosphere before they were trypsinized and analyzed byfluorescence microscopy (A) and flow cytometry (B) as shown in FIG. 5 ;A) show green fluorescence signals merged with phase contrast images.Histograms in B) show the green fluorescence intensity on a logarithmicscale on the x-axis and the normalized frequency on the y-axis. 1: HuH7cells not treated with any antibody or EVs. 2: HuH7 cells treated withcontrol EVs which were incubated with anti-Lamin B2 antibodies. 3: HuH7cells treated with Fc-binding EVs which were incubated with anti-LaminB2 antibodies. FIG. 5 shows that signals of fluorescent antibodies areclearly present in cells treated with Fc-binding EVs plus antibody,while fluorescence signals are absent in untreated (1) or control EVtreated (2). This demonstrates that antibodies can be deliveredintracellularly via Fc-binding EVs, and that binding to EVs dramaticallyincreases uptake of antibodies into cells.

To demonstrate functional intracellular delivery, anti-NFkB antibodies(anti-NFkB-Ab) were incubated with respective EVs for 1 h at 37° C. Areporter cell line, HEK cells stably expressing NFkB-luciferase, weretreated with 5 ng/ml hTNF-alpha and the EV-Ab-mix. After 6 hours oftreatment the luciferase activity was measured. FIG. 6 shows successfulinhibition when the anti-NFkB-ab is delivered by Fc-binding EVs.

1-23. (canceled)
 24. Use of an extracellular vesicle (EV), comprising atleast one Fc binding polypeptide, as a research tool, a diagnostic tool,an imaging tool, as a biological reference material (RM), as anexperimental control or an experimental standard.
 25. The use accordingto claim 24, wherein said use is in flow cytometry, imaging flowcytometry, nanoparticle tracking analysis, nanoimaging, confocalmicroscopy, fluorescence microscopy, dynamic light scattering, electronmicroscopy, luminescence-based detection, fluorescence correlationspectroscopy, fluorescence resonance energy transfer (FRET)-basedtechniques, positive or negative selection techniques based on magnetic,fluorescent, or buoyancy-based labelling, positive or negative selectiontechniques based on particle density, charge or size, or any combinationthereof.
 26. The use according to claim 24, wherein the at least one Fcbinding polypeptide forms part of a fusion protein together with atleast one exosomal polypeptide.
 27. The use according to claim 24,wherein the Fc binding polypeptide is bound to the Fc domain of an Fccontaining protein.
 28. The use according to claim 25, wherein the Fccontaining protein is an antibody.
 29. The use according to claim 25,wherein the Fc containing protein is attached to at least onefluorophore, at least one radioactive label, at least one PET ligand, atleast one MRI agent, a nanoparticle, an inorganic compound, or any otherdetection moiety.
 30. The use according to claim 24, wherein the use isas a biological RM for flow cytometry, nanoparticle tracking analysis,or dynamic light scattering.
 31. The use according to claim 30, whereinthe RM is used for compensation, calibration, standardization,validation, or any other similar purpose.
 32. The use according to claim24, wherein the EV itself comprises at least one detection moiety. 33.The use according to claim 32, wherein the detection moiety is selectedfrom the group comprising at least one fluorophore, at least one dye, atleast one fluorescent polypeptide, at least one radioactive label, atleast one PET agent, at least one MRI agent, an inorganic compound, anorganic compound, or any other detection moiety.
 34. An in vitro, invivo or ex vivo method for delivery of an Fc containing protein to atarget cell, comprising (i) providing an EV comprising at least one Fcbinding polypeptide, (ii) allowing the Fc binding polypeptide to bindthe Fc containing protein, (iii) contacting a target cell with the EVhaving attached to its Fc binding polypeptide the Fc containing protein.35. The method according to claim 34, wherein the Fc containing proteinis delivered into the intracellular environment of a target cell. 36.The method according to claim 34, wherein the at least one Fc bindingpolypeptide forms part of a fusion protein together with at least oneexosomal polypeptide.
 37. The method according to claim 34, wherein theFc containing protein is an antibody.
 38. The method according to claim34, wherein the Fc containing protein is attached to at least onefluorophore, at least one radioactive label, at least one PET ligand, atleast one MRI agent, a nanoparticle, an inorganic compound, or any otherdetection moiety.
 39. The method according to claim 34, wherein the EVitself comprises a detection moiety, optionally selected from the groupcomprising at least one fluorophore, at least one dye, at least onefluorescent polypeptide, at least one radioactive label, at least onePET agent, at least one MM agent, a nanoparticle, an inorganic compound,or any other detection moiety.
 40. A detection kit comprising an EV,wherein the EV comprises at least one fusion protein comprising at leastone Fc binding polypeptide and at least one exosomal polypeptide. 41.The detection kit according to claim 40, wherein the at least one Fcbinding polypeptide is bound to the Fc domain of an Fc containingprotein.
 42. The detection kit according to claim 40, wherein the Fccontaining protein is an antibody.
 43. The detection kit according toclaim 40, wherein the Fc containing protein is attached to at least onefluorophore, at least one radioactive label, at least one PET ligand, atleast one MRI agent, a nanoparticle, an inorganic compound, or any otherdetection moiety.
 44. The detection kit according to claim 40, for usein the detection of an extracellular or intracellular biomolecule. 45.The detection kit according to claim 44, wherein the detection of thebiomolecule takes place in live or dead cells or tissues or organs. 46.The detection kit according to claim 44, wherein the detection of thebiomolecule takes place in vitro, ex vivo or in vivo.
 47. The detectionkit according to claim 40, wherein the EV itself comprises at least oneadditional detection moiety, optionally selected from at least onefluorophore such as at least one fluorescent polypeptide, at least onepolypeptide producing bioluminescence, at least one radioactive label,at least one PET agent, at least one MRI agent, a nanoparticle, aninorganic compound, or any other detection moiety.
 48. The detection kitaccording to claim 40, for use in flow cytometry, nanoparticle trackinganalysis, nanoimaging, confocal microscopy, fluorescence microscopy,dynamic light scattering, electron microscopy, luminescence-baseddetection, fluorescence correlation spectroscopy, fluorescence resonanceenergy transfer (FRET)-based techniques, positive or negative selectiontechniques based on magnetic, fluorescent, or buoyancy-based labelling,positive or negative selection techniques based on density, charge orsize, or any combination thereof.
 49. A kit of parts comprising (i) anEV comprising at least one Fc binding polypeptide, and (ii) at least oneFc containing protein.
 50. The kit of parts according to claim 49,wherein the at least one Fc binding polypeptide forms part of a fusionprotein together with at least one exosomal polypeptide.
 51. The kit ofparts according to claim 49, wherein the Fc containing protein is anantibody.
 52. The kit of parts according to claim 49, wherein the Fccontaining protein is attached to at least one fluorophore, at least oneradioactive label, at least one PET agent, at least one MRI agent, ananoparticle, an inorganic compound, or any other detection moiety. 53.The kit of parts according to claim 49, wherein the EV comprises atleast one additional detection moiety selected from at least onefluorophore, at least one radioactive label, at least one PET ligand, atleast one MRI agent, a nanoparticle, an inorganic compound, or any otherdetection moiety.
 54. A nanoparticle complex between (i) an EVcomprising at least one Fc binding polypeptide and (ii) an Fc containingprotein.
 55. The nanoparticle complex according to claim 54, wherein theat least one Fc binding polypeptide forms part of a fusion proteintogether with at least one exosomal polypeptide.
 56. The nanoparticlecomplex according to claim 54, wherein the Fc containing protein is anantibody.
 57. The nanoparticle complex according to claim 54, whereinthe Fc containing protein is attached to at least one fluorophore, atleast one radioactive label, at least one PET agent, at least one MMagent, a nanoparticle, an inorganic compound, or any other detectionmoiety.
 58. The nanoparticle complex according to claim 54 for use in:(a) flow cytometry, nanoparticle tracking analysis, nanoimaging,confocal microscopy, fluorescence microscopy, dynamic light scattering,electron microscopy, luminescence-based detection, fluorescencecorrelation spectroscopy, fluorescence resonance energy transfer(FRET)-based techniques, positive or negative selection techniques basedon magnetic, fluorescent, or buoyancy-based labelling, positive ornegative selection techniques based on density, charge or size, (b) as aresearch tool, an imaging tool, a diagnostic tool, an imaging tool, areference material, a detection kit, or (c) for in vitro, in vivo or exvivo delivery of an Fc containing protein.
 59. Use of a detection kitaccording to claim 40, (a) in flow cytometry, nanoparticle trackinganalysis, nanoimaging, confocal microscopy, fluorescence microscopy,dynamic light scattering, electron microscopy, luminescence-baseddetection, fluorescence correlation spectroscopy, fluorescence resonanceenergy transfer (FRET)-based techniques, positive or negative selectiontechniques based on magnetic, fluorescent, or buoyancy-based labelling,positive or negative selection techniques based on density, charge orsize, (b) as a research tool, a diagnostic tool, an imaging tool, abiological RM, a detection kit, or (c) for in vitro, in vivo, or ex vivodelivery of an Fc containing protein or any combination thereof.